2024-03-28T14:25:51Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/3113092019-08-30T11:33:55Zcom_10033_311308col_10033_559591
An enzyme from Auricularia auricula-judae combining both benzoyl and cinnamoyl esterase activity
Haase-Aschoff, Paul
Linke, Diana
Nimtz, Manfred
Popper, Lutz
Berger, Ralf G.
Dept. of structure and function of proteins, Helmhotz Centre for infection research, D-38124 Braunschweig, Germany
2014-01-14T13:14:20Z
2014-01-14T13:14:20Z
2014-01-14T13:14:20Z
2014-01-14
Article
An enzyme from Auricularia auricula-judae combining both benzoyl and cinnamoyl esterase activity 2013, 48 (12):1872 Process Biochemistry
13595113
10.1016/j.procbio.2013.09.016
http://hdl.handle.net/10033/311309
Process Biochemistry
http://linkinghub.elsevier.com/retrieve/pii/S1359511313005497
Archived with thanks to Process Biochemistry
oai:repository.helmholtz-hzi.de:10033/3260092019-08-30T11:36:05Zcom_10033_311308col_10033_559591
Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD.
Vit, Allegra
Misson, Laëtitia
Blankenfeldt, Wulf
Seebeck, Florian Peter
Dept of structure and functions of proteins, Hemholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany.
Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit.
2014-09-09T13:48:46Z
2014-09-09T13:48:46Z
2014-09-09T13:48:46Z
2014-05
Article
Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD. 2014, 70 (Pt 5):676-80 Acta Crystallogr F Struct Biol Commun
2053-230X
24817736
10.1107/S2053230X1400805X
http://hdl.handle.net/10033/326009
Acta crystallographica. Section F, Structural biology communications
en
Archived with thanks to Acta crystallographica. Section F, Structural biology communications
oai:repository.helmholtz-hzi.de:10033/3370282019-08-30T11:34:48Zcom_10033_311308col_10033_559591
The structural biology of phenazine biosynthesis.
Blankenfeldt, Wulf
Parsons, James F
Helmholtz Centre for Infection Research, Structure and Function of Proteins, Inhoffenstr. 7, 38124 Braunschweig, Germany. Electronic address: wulf.blankenfeldt@helmholtz-hzi.de.
The phenazines are a class of over 150 nitrogen-containing aromatic compounds of bacterial and archeal origin. Their redox properties not only explain their activity as broad-specificity antibiotics and virulence factors but also enable them to function as respiratory pigments, thus extending their importance to the primary metabolism of phenazine-producing species. Despite their discovery in the mid-19th century, the molecular mechanisms behind their biosynthesis have only been unraveled in the last decade. Here, we review the contribution of structural biology that has led to our current understanding of phenazine biosynthesis.
2014-12-10T15:09:03Z
2014-12-10T15:09:03Z
2014-12-10T15:09:03Z
2014-09-09
Article
The structural biology of phenazine biosynthesis. 2014, 29C:26-33 Curr. Opin. Struct. Biol.
1879-033X
25215885
10.1016/j.sbi.2014.08.013
http://hdl.handle.net/10033/337028
Current opinion in structural biology
oai:repository.helmholtz-hzi.de:10033/3465282019-08-30T11:37:23Zcom_10033_311308col_10033_559591
Oligomerization inhibits Legionella pneumophila PlaB phospholipase A activity.
Kuhle, Katja
Krausze, Joern
Curth, Ute
Rössle, Manfred
Heuner, Klaus
Lang, Christina
Flieger, Antje
The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity within the PlaB C terminus, and the significance of PlaB-derived lipolytic activity for L. pneumophila intracellular replication. We determined by means of analytical ultracentrifugation and small angle x-ray scattering analysis that PlaB forms homodimers and homotetramers. The C-terminal 5, 10, or 15 amino acids, although the individual regions contributed to PLA activity, were not essential for protein tetramerization. Infection of mouse macrophages with L. pneumophila wild type, plaB knock-out mutant, and plaB complementing or various mutated plaB-harboring strains showed that catalytic activity of PlaB promotes intracellular replication. We observed that PlaB was most active in the lower nanomolar concentration range but not at or only at a low level at concentration above 0.1 μm where it exists in a dimer/tetramer equilibrium. We therefore conclude that PlaB is a virulence factor that, on the one hand, assembles in inactive tetramers at micromolar concentrations. On the other hand, oligomer dissociation at nanomolar concentrations activates PLA activity. Our data highlight the first example of concentration-dependent phospholipase inactivation by tetramerization, which may protect the bacterium from internal PLA activity, but enzyme dissociation may allow its activation after export.
2015-03-11T14:54:36Z
2015-03-11T14:54:36Z
2015-03-11T14:54:36Z
2014-07-04
Article
Oligomerization inhibits Legionella pneumophila PlaB phospholipase A activity. 2014, 289 (27):18657-66 J. Biol. Chem.
1083-351X
24811180
10.1074/jbc.M114.573196
http://hdl.handle.net/10033/346528
The Journal of biological chemistry
en
oai:repository.helmholtz-hzi.de:10033/5566622019-08-30T11:26:42Zcom_10033_311308col_10033_559591
The immunoglobulin M-degrading enzyme of Streptococcus suis, Ide Ssuis , is involved in complement evasion.
Seele, Jana
Beineke, Andreas
Hillermann, Lena-Maria
Jaschok-Kentner, Beate
von Pawel-Rammingen, Ulrich
Valentin-Weigand, Peter
Baums, Christoph Georg
Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany.
Streptococcus (S.) suis is one of the most important pathogens in pigs causing meningitis, arthritis, endocarditis and serositis. Furthermore, it is also an emerging zoonotic agent. In our previous work we identified a highly specific IgM protease in S. suis, designated Ide Ssuis . The objective of this study was to characterize the function of Ide Ssuis in the host-pathogen interaction. Edman-sequencing revealed that Ide Ssuis cleaves the heavy chain of the IgM molecule between constant domain 2 and 3. As the C1q binding motif is located in the C3 domain, we hypothesized that Ide Ssuis is involved in complement evasion. Complement-mediated hemolysis induced by porcine hyperimmune sera containing erythrocyte-specific IgM was abrogated by treatment of these sera with recombinant Ide Ssuis . Furthermore, expression of Ide Ssuis reduced IgM-triggered complement deposition on the bacterial surface. An infection experiment of prime-vaccinated growing piglets suggested attenuation in the virulence of the mutant 10Δide Ssuis . Bactericidal assays confirmed a positive effect of Ide Ssuis expression on bacterial survival in porcine blood in the presence of high titers of specific IgM. In conclusion, this study demonstrates that Ide Ssuis is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.
2015-06-10T14:13:04Z
2015-06-10T14:13:04Z
2015-06-10T14:13:04Z
2015
Article
The immunoglobulin M-degrading enzyme of Streptococcus suis, Ide Ssuis , is involved in complement evasion. 2015, 46 (1):45 Vet. Res.
1297-9716
25928761
10.1186/s13567-015-0171-6
http://hdl.handle.net/10033/556662
Veterinary research
en
eu-repo/grantAgreement/EC/FP7/281473
openAccess
oai:repository.helmholtz-hzi.de:10033/5613172019-08-30T11:25:43Zcom_10033_311308col_10033_559591
Crystallization, room-temperature X-ray diffraction and preliminary analysis of Kaposi's sarcoma herpesvirus LANA bound to DNA.
Hellert, Jan
Krausze, Joern
Schulz, Thomas F
Lührs, Thorsten
Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany.
The latency-associated nuclear antigen (LANA) is the latent origin-binding protein and chromatin anchor of the Kaposi's sarcoma herpesvirus (KSHV/HHV-8) genome. Its C-terminal domain (CTD) binds sequence-specifically to the viral origin of replication, whereas the N-terminal domain links it to nucleosomes of cellular chromatin for long-term persistence in dividing host cells. Here, the crystallization and X-ray data acquisition of a mutant LANA CTD in complex with its wild-type target DNA LBS1 is described. This report describes the rational protein engineering for successful co-crystallization with DNA and X-ray diffraction data collection at room temperature on the high-brilliance third-generation synchrotron PETRA III at DESY, Germany.
2015-07-31T14:30:59Z
2015-07-31T14:30:59Z
2015-07-31T14:30:59Z
2014-11
Article
Crystallization, room-temperature X-ray diffraction and preliminary analysis of Kaposi's sarcoma herpesvirus LANA bound to DNA. 2014, 70 (Pt 11):1570-4 Acta Crystallogr F Struct Biol Commun
2053-230X
25372834
10.1107/S2053230X14019906
http://hdl.handle.net/10033/561317
Acta crystallographica. Section F, Structural biology communications
en
oai:repository.helmholtz-hzi.de:10033/6207882019-08-30T11:36:32Zcom_10033_311308col_10033_559591
Side effects of chaperone gene co-expression in recombinant protein production
Martínez-Alonso, Mónica
García-Fruitós, Elena
Ferrer-Miralles, Neus
Rinas, Ursula
Villaverde, Antonio
Abstract Insufficient availability of molecular chaperones is observed as a major bottleneck for proper protein folding in recombinant protein production. Therefore, co-production of selected sets of cell chaperones along with foreign polypeptides is a common approach to increase the yield of properly folded, recombinant proteins in bacterial cell factories. However, unbalanced amounts of folding modulators handling folding-reluctant protein species might instead trigger undesired proteolytic activities, detrimental regarding recombinant protein stability, quality and yield. This minireview summarizes the most recent observations of chaperone-linked negative side effects, mostly focusing on DnaK and GroEL sets, when using these proteins as folding assistant agents. These events are discussed in the context of the complexity of the cell quality network and the consequent intricacy of the physiological responses triggered by protein misfolding.
2017-01-27T11:49:56Z
2017-01-27T11:49:56Z
2017-01-27T11:49:56Z
2010-09-02
Journal Article
Microbial Cell Factories. 2010 Sep 02;9(1):64
http://dx.doi.org/10.1186/1475-2859-9-64
http://hdl.handle.net/10033/620788
en
Martínez-Alonso et al.
oai:repository.helmholtz-hzi.de:10033/6207852019-08-30T11:37:44Zcom_10033_311308col_10033_559591
High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells
Jäger, Volker
Büssow, Konrad
Wagner, Andreas
Weber, Susanne
Hust, Michael
Frenzel, André
Schirrmann, Thomas
Abstract Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. Results In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Conclusion Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.
2017-01-27T11:45:00Z
2017-01-27T11:45:00Z
2017-01-27T11:45:00Z
2013-06-26
Journal Article
BMC Biotechnology. 2013 Jun 26;13(1):52
http://dx.doi.org/10.1186/1472-6750-13-52
http://hdl.handle.net/10033/620785
en
Jäger et al.; licensee BioMed Central Ltd.
oai:repository.helmholtz-hzi.de:10033/6207792019-08-30T11:25:43Zcom_10033_311308col_10033_559591
Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx
Wilke, Sonja
Krausze, Joern
Büssow, Konrad
Abstract Background The family of lysosome-associated membrane proteins (LAMP) comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68). LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of the glycoconjugate coat present on the inside of the lysosomal membrane, the 'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain of 150 to 200 amino acids with two disulfide bonds. Results The crystal structure of the conserved domain of human DC-LAMP was solved. It is the first high-resolution structure of a heavily glycosylated lysosomal membrane protein. The structure represents a novel β-prism fold formed by two β-sheets bent by β-bulges and connected by a disulfide bond. Flexible loops and a hydrophobic pocket represent possible sites of molecular interaction. Computational models of the glycosylated luminal regions of LAMP-1 and LAMP-2 indicate that the proteins adopt a compact conformation in close proximity to the lysosomal membrane. The models correspond to the thickness of the lysosomal glycoprotein coat of only 5 to 12 nm, according to electron microscopy. Conclusion The conserved luminal domain of lysosome-associated membrane proteins forms a previously unknown β-prism fold. Insights into the structure of the lysosomal glycoprotein coat were obtained by computational models of the LAMP-1 and LAMP-2 luminal regions.
2017-01-27T10:49:29Z
2017-01-27T10:49:29Z
2017-01-27T10:49:29Z
2012-07-19
Journal Article
BMC Biology. 2012 Jul 19;10(1):62
http://dx.doi.org/10.1186/1741-7007-10-62
http://hdl.handle.net/10033/620779
en
Wilke et al; licensee BioMed Central Ltd.
oai:repository.helmholtz-hzi.de:10033/6205592019-08-30T11:27:16Zcom_10033_311308col_10033_559591
Crystal structure of AibC, a reductase involved in alternative de novo isovaleryl coenzyme A biosynthesis in Myxococcus xanthus.
Bock, Tobias
Müller, Rolf
Blankenfeldt, Wulf
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Isovaleryl coenzyme A (IV-CoA) performs a crucial role during development and fruiting-body formation in myxobacteria, which is reflected in the existence of a de novo biosynthetic pathway that is highly upregulated when leucine, the common precursor of IV-CoA, is limited. The final step in de novo IV-CoA biosynthesis is catalyzed by AibC, a medium-chain dehydrogenase/reductase. Here, the crystal structure of AibC from Myxococcus xanthus refined to 2.55 Å resolution is presented. The protein adopts two different conformations in the crystal lattice, which is a consequence of partial interaction with the purification tag. Based on this structure, it is suggested that AibC most probably uses a Zn(2+)-supported catalytic mechanism in which NADPH is preferred over NADH. Taken together, this study reveals structural details of the alternative IV-CoA-producing pathway in myxobacteria, which may serve as a base for further biotechnological research and biofuel production.
2016-10-20T09:42:10Z
2016-10-20T09:42:10Z
2016-10-20T09:42:10Z
2016-08
Article
Crystal structure of AibC, a reductase involved in alternative de novo isovaleryl coenzyme A biosynthesis in Myxococcus xanthus. 2016, 72 (Pt 8):652-8 Acta Crystallogr F Struct Biol Commun
2053-230X
27487931
10.1107/S2053230X16011146
http://hdl.handle.net/10033/620559
Acta crystallographica. Section F, Structural biology communications
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6206882019-08-30T11:33:57Zcom_10033_311308com_10033_620618col_10033_620619col_10033_559591col_10033_620561
The AibR-isovaleryl coenzyme A regulator and its DNA binding site - a model for the regulation of alternative de novo isovaleryl coenzyme A biosynthesis in Myxococcus xanthus.
Bock, Tobias
Volz, Carsten
Hering, Vanessa
Scrima, Andrea
Müller, Rolf
Blankenfeldt, Wulf
Hel,holtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Isovaleryl coenzyme A (IV-CoA) is an important building block of iso-fatty acids. In myxobacteria, IV-CoA is essential for the formation of signaling molecules involved in fruiting body formation. Leucine degradation is the common source of IV-CoA, but a second, de novo biosynthetic route to IV-CoA termed AIB (alternative IV-CoA biosynthesis) was recently discovered in M. xanthus The AIB-operon contains the TetR-like transcriptional regulator AibR, which we characterize in this study. We demonstrate that IV-CoA binds AibR with micromolar affinity and show by gelshift experiments that AibR interacts with the promoter region of the AIB-operon once IV-CoA is present. We identify an 18-bp near-perfect palindromic repeat as containing the AibR operator and provide evidence that AibR also controls an additional genomic locus coding for a putative acetyl-CoA acetyltransferase. To elucidate atomic details, we determined crystal structures of AibR in the apo, the IV-CoA- and the IV-CoA-DNA-bound state to 1.7 Å, 2.35 Å and 2.92 Å, respectively. IV-CoA induces partial unfolding of an α-helix, which allows sequence-specific interactions between AibR and its operator. This study provides insights into AibR-mediated regulation and shows that AibR functions in an unusual TetR-like manner by blocking transcription not in the ligand-free but in the effector-bound state.
2017-01-09T15:31:16Z
2017-01-09T15:31:16Z
2017-01-09T15:31:16Z
2016-12-09
Article
The AibR-isovaleryl coenzyme A regulator and its DNA binding site - a model for the regulation of alternative de novo isovaleryl coenzyme A biosynthesis in Myxococcus xanthus. 2016 Nucleic Acids Res.
1362-4962
27940564
10.1093/nar/gkw1238
http://hdl.handle.net/10033/620688
Nucleic acids research
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6209272019-08-30T11:35:39Zcom_10033_311308com_10033_620618col_10033_620619col_10033_620619col_10033_559591
Crystal Structure of the HMG-CoA Synthase MvaS from the Gram-Negative Bacterium Myxococcus xanthus.
Bock, Tobias
Kasten, Janin
Müller, Rolf
Blankenfeldt, Wulf
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
A critical step in bacterial isoprenoid production is the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) catalyzed by HMG-CoA synthase (HMGCS). In myxobacteria, this enzyme is also involved in a recently discovered alternative and acetyl-CoA-dependent isovaleryl CoA biosynthesis pathway. Here we present crystal structures of MvaS, the HMGCS from Myxococcus xanthus, in complex with CoA and acetylated active site Cys115, with the second substrate acetoacetyl CoA and with the product of the condensation reaction, 3-hydroxy-3-methylglutaryl CoA. With these structures, we show that MvaS uses the common HMGCS enzymatic mechanism and provide evidence that dimerization plays a role in the formation and stability of the active site. Overall, MvaS shows features typical of the eukaryotic HMGCS and exhibits differences from homologues from Gram-positive bacteria. This study provides insights into myxobacterial alternative isovaleryl CoA biosynthesis and thereby extends the toolbox for the biotechnological production of renewable fuel and chemicals.
2017-05-22T14:01:14Z
2017-05-22T14:01:14Z
2017-05-22T14:01:14Z
2016-07-01
Article
Crystal Structure of the HMG-CoA Synthase MvaS from the Gram-Negative Bacterium Myxococcus xanthus. 2016, 17 (13):1257-62 Chembiochem
1439-7633
27124816
10.1002/cbic.201600070
http://hdl.handle.net/10033/620927
Chembiochem : a European journal of chemical biology
en
oai:repository.helmholtz-hzi.de:10033/6209282021-07-06T11:57:21Zcom_10033_311308com_10033_622921col_10033_622925col_10033_559591
Intrabodies against the Polysialyltransferases ST8SiaII and ST8SiaIV inhibit Polysialylation of NCAM in rhabdomyosarcoma tumor cells.
Somplatzki, Stefan
Mühlenhoff, Martina
Kröger, Andrea
Gerardy-Schahn, Rita
Böldicke, Thomas
Helmholtz Centr for infection research
Polysialic acid (polySia) is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis. Polysialylation of NCAM is mediated by two Golgi-resident polysialyltransferases (polyST) ST8SiaII and ST8SiaIV. Intracellular antibodies (intrabodies; IB) expressed inside the ER and retaining proteins passing the ER such as cell surface receptors or secretory proteins provide an efficient means of protein knockdown. To inhibit the function of ST8SiaII and ST8SiaIV specific ER IBs were generated starting from two corresponding hybridoma clones. Both IBs αST8SiaII-IB and αST8SiaIV-IB were constructed in the scFv format and their functions characterized in vitro and in vivo.
2017-05-24T12:59:17Z
2017-05-24T12:59:17Z
2017-05-24T12:59:17Z
2017-05-12
Article
Intrabodies against the Polysialyltransferases ST8SiaII and ST8SiaIV inhibit Polysialylation of NCAM in rhabdomyosarcoma tumor cells. 2017, 17 (1):42 BMC Biotechnol.
1472-6750
28499450
10.1186/s12896-017-0360-7
http://hdl.handle.net/10033/620928
BMC biotechnology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6210412020-08-05T10:54:26Zcom_10033_311308col_10033_559591
Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF.
Diederich, Christina
Leypold, Mario
Culka, Martin
Weber, Hansjörg
Breinbauer, Rolf
Ullmann, G Matthias
Blankenfeldt, Wulf
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Phenazines are bacterial virulence and survival factors with important roles in infectious disease. PhzF catalyzes a key reaction in their biosynthesis by isomerizing (2 S,3 S)-2,3-dihydro-3-hydroxy anthranilate (DHHA) in two steps, a [1,5]-hydrogen shift followed by tautomerization to an aminoketone. While the [1,5]-hydrogen shift requires the conserved glutamate E45, suggesting acid/base catalysis, it also shows hallmarks of a sigmatropic rearrangement, namely the suprafacial migration of a non-acidic proton. To discriminate these mechanistic alternatives, we employed enzyme kinetic measurements and computational methods. Quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the activation barrier of a proton shuttle mechanism involving E45 is significantly lower than that of a sigmatropic [1,5]-hydrogen shift. QM/MM also predicted a large kinetic isotope effect, which was indeed observed with deuterated substrate. For the tautomerization, QM/MM calculations suggested involvement of E45 and an active site water molecule, explaining the observed stereochemistry. Because these findings imply that PhzF can act only on a limited substrate spectrum, we also investigated the turnover of DHHA derivatives, of which only O-methyl and O-ethyl DHHA were converted. Together, these data reveal how PhzF orchestrates a water-free with a water-dependent step. Its unique mechanism, specificity and essential role in phenazine biosynthesis may offer opportunities for inhibitor development.
2017-08-04T07:58:49Z
2017-08-04T07:58:49Z
2017-08-04T07:58:49Z
2017-07-24
Article
Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF. 2017, 7 (1):6272 Sci Rep
2045-2322
28740244
10.1038/s41598-017-06278-w
http://hdl.handle.net/10033/621041
Scientific reports
en
560678
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6211072019-08-30T11:27:16Zcom_10033_311624com_10033_6839com_10033_311308col_10033_311625col_10033_559591
Structural, mechanistic and functional insight into gliotoxin bis-thiomethylation in Aspergillus fumigatus.
Dolan, Stephen K
Bock, Tobias
Hering, Vanessa
Owens, Rebecca A
Jones, Gary W
Blankenfeldt, Wulf
Doyle, Sean
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus Self-resistance against gliotoxin is effected by the gliotoxin oxidase GliT, and attenuation of gliotoxin biosynthesis is catalysed by gliotoxin S-methyltransferase GtmA. Here we describe the X-ray crystal structures of GtmA-apo (1.66 Å), GtmA complexed to S-adenosylhomocysteine (1.33 Å) and GtmA complexed to S-adenosylmethionine (2.28 Å), providing mechanistic insights into this important biotransformation. We further reveal that simultaneous elimination of the ability of A. fumigatus to dissipate highly reactive dithiol gliotoxin, via deletion of GliT and GtmA, results in the most significant hypersensitivity to exogenous gliotoxin observed to date. Indeed, quantitative proteomic analysis of ΔgliT::ΔgtmA reveals an uncontrolled over-activation of the gli-cluster upon gliotoxin exposure. The data presented herein reveal, for the first time, the extreme risk associated with intracellular dithiol gliotoxin biosynthesis-in the absence of an efficient dismutation capacity. Significantly, a previously concealed protective role for GtmA and functionality of ETP bis-thiomethylation as an ancestral protection strategy against dithiol compounds is now evident.
2017-09-13T13:49:00Z
2017-09-13T13:49:00Z
2017-09-13T13:49:00Z
2017-02
Article
Structural, mechanistic and functional insight into gliotoxin bis-thiomethylation in Aspergillus fumigatus. 2017, 7 (2) Open Biol
2046-2441
28179499
10.1098/rsob.160292
http://hdl.handle.net/10033/621107
Open biology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6211372019-08-30T11:34:48Zcom_10033_311308col_10033_559591
Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain.
DiCara, Danielle M
Chirgadze, Dimitri Y
Pope, Anthony R
Karatt-Vellatt, Aneesh
Winter, Anja
Slavny, Peter
van den Heuvel, Joop
Parthiban, Kothai
Holland, Jane
Packman, Len C
Mavria, Georgia
Hoffmann, Jens
Birchmeier, Walter
Gherardi, Ermanno
McCafferty, John
Helmholtz-Zentrum für Infektionsforschung GmbH. Inhoffenstr. 7, 38124 Braunschweig, Germany.
The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the "compact", InternalinB-bound conformation, but not when MET is in the "open" conformation. These findings provide further support for the importance of the "compact" conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.
2017-10-13T13:21:14Z
2017-10-13T13:21:14Z
2017-10-13T13:21:14Z
2017-08-21
Article
Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain. 2017, 7 (1):9000 Sci Rep
2045-2322
28827556
10.1038/s41598-017-09460-2
http://hdl.handle.net/10033/621137
Scientific reports
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6211452019-08-30T11:33:24Zcom_10033_311308col_10033_559591
ER-targeted intrabodies mediating specific in vivo knockdown of transitory proteins in comparison to RNAi
Backhaus, Oliver
Böldicke, Thomas
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
In animals and mammalian cells, protein function can be analyzed by nucleotide sequence-based methods such as gene knockout, targeted gene disruption, CRISPR/Cas, TALEN, zinc finger nucleases, or the RNAi technique. Alternatively, protein knockdown approaches are available based on direct interference of the target protein with the inhibitor.Among protein knockdown techniques, the endoplasmic reticulum (ER) intrabodies arepotent molecules for protein knockdown in vitro and in vivo. These molecules are increasingly used for protein knockdown in living cells and transgenic mice. ER intrabody knockdown technique is based on the retention of membrane proteins and secretory proteins inside the ER, mediated by recombinant antibody fragments. In contrast to nucleotide sequence-based methods, the intrabody-mediated knockdown actsonly on the posttranslational level. In this review, the ER intrabody technology has been compared with the RNAi technique on the molecular level. The generation of intrabodies and RNAi has also been discussed. Specificity and off-target effects (OTE) of these molecules as well as the therapeutic potential of ER intrabodies and RNAi have been compared.
2017-10-25T09:49:59Z
2017-10-25T09:49:59Z
2017-10-25T09:49:59Z
2017-10-25
Book chapter
Oliver Backhaus and Thomas Böldicke (April 6th 2016). ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi, RNA Interference Ibrokhim Y. Abdurakhmonov, IntechOpen, DOI: 10.5772/62103. Available from: https://www.intechopen.com/books/rna-interference/er-targeted-intrabodies-mediating-specific-in-vivo-knockdown-of-transitory-proteins-in-comparison-to
978-953-51-2272-2
10.5772/62103
http://hdl.handle.net/10033/621145
https://www.intechopen.com/books/rna-interference/er-targeted-intrabodies-mediating-specific-in-vivo-knockdown-of-transitory-proteins-in-comparison-to
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6211842019-08-30T11:25:11Zcom_10033_620533com_10033_311308com_10033_620618col_10033_620534col_10033_620619col_10033_559591
Biosynthesis of methyl-proline containing griselimycins, natural products with anti-tuberculosis activity.
Lukat, Peer
Katsuyama, Yohei
Wenzel, Silke
Binz, Tina
König, Claudia
Blankenfeldt, Wulf
Brönstrup, Mark
Müller, Rolf
Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany.
Griselimycins (GMs) are depsidecapeptides with superb anti-tuberculosis activity. They contain up to three (2S,4R)-4-methyl-prolines (4-MePro), of which one blocks oxidative degradation and increases metabolic stability in animal models. The natural congener with this substitution is only a minor component in fermentation cultures. We showed that this product can be significantly increased by feeding the reaction with 4-MePro and we investigated the molecular basis of 4-MePro biosynthesis and incorporation. We identified the GM biosynthetic gene cluster as encoding a nonribosomal peptide synthetase and a sub-operon for 4-MePro formation. Using heterologous expression, gene inactivation, and in vitro experiments, we showed that 4-MePro is generated by leucine hydroxylation, oxidation to an aldehyde, and ring closure with subsequent reduction. The crystal structures of the leucine hydroxylase GriE have been determined in complex with substrates and products, providing insight into the stereospecificity of the reaction.
2017-11-27T15:04:00Z
2017-11-27T15:04:00Z
2017-11-27T15:04:00Z
2017-11-01
Article
Biosynthesis of methyl-proline containing griselimycins, natural products with anti-tuberculosis activity. 2017, 8 (11):7521-7527 Chem Sci
2041-6520
29163906
10.1039/c7sc02622f
http://hdl.handle.net/10033/621184
Chemical science
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6213192019-08-30T11:25:11Zcom_10033_311308col_10033_559591
Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum.
Füller, Janis J
Röpke, René
Krausze, Joern
Rennhack, Kim E
Daniel, Nils P
Blankenfeldt, Wulf
Schulz, Stefan
Jahn, Dieter
Moser, Jürgen
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Violacein is a natural purple pigment of Chromobacterium violaceum with potential medical applications as antimicrobial, antiviral, and anticancer drugs. The initial step of violacein biosynthesis is the oxidative conversion of l-tryptophan into the corresponding α-imine catalyzed by the flavoenzyme l-tryptophan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-methylpropanoic acid) and a product-related (2-(1H-indol-3-ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was synthesized for subsequent kinetic and x-ray crystallographic investigations. Structures of the binary VioA·FADH2 and of the ternary VioA·FADH2·2-(1H-indol-3-ylmethyl)prop-2-enoic acid complex were resolved. VioA forms a "loosely associated" homodimer as indicated by small-angle x-ray scattering experiments. VioA belongs to the glutathione reductase family 2 of FAD-dependent oxidoreductases according to the structurally conserved cofactor binding domain. The substrate-binding domain of VioA is mainly responsible for the specific recognition of l-tryptophan. Other canonical amino acids were efficiently discriminated with a minor conversion of l-phenylalanine. Furthermore, 7-aza-tryptophan, 1-methyl-tryptophan, 5-methyl-tryptophan, and 5-fluoro-tryptophan were efficient substrates of VioA. The ternary product-related VioA structure indicated involvement of protein domain movement during enzyme catalysis. Extensive structure-based mutagenesis in combination with enzyme kinetics (using l-tryptophan and substrate analogs) identified Arg(64), Lys(269), and Tyr(309) as key catalytic residues of VioA. An increased enzyme activity of protein variant H163A in the presence of l-phenylalanine indicated a functional role of His(163) in substrate binding. The combined structural and mutational analyses lead to the detailed understanding of VioA substrate recognition. Related strategies for the in vivo synthesis of novel violacein derivatives are discussed.
2018-03-12T13:07:45Z
2018-03-12T13:07:45Z
2018-03-12T13:07:45Z
2016
Article
Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum. 2016, 291 (38):20068-84 J. Biol. Chem.
1083-351X
27466367
10.1074/jbc.M116.741561
http://hdl.handle.net/10033/621319
The Journal of biological chemistry
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6214442019-08-22T12:38:05Zcom_10033_311308col_10033_559591
Single domain antibodies for the knockdown of cytosolic and nuclear proteins.
Böldicke, Thomas
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
camelid VHHs
cytosolic/nuclear intrabodies
human VH
human VL
intrabodies
scFv fragment
shark vNARs
single domain antibodies
Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.
2018-08-08T14:16:46Z
2018-08-08T14:16:46Z
2018-08-08T14:16:46Z
2017-05-01
Article
1469-896X
28271570
10.1002/pro.3154
http://hdl.handle.net/10033/621444
https://www.ncbi.nlm.nih.gov/pmc/?term=5405437
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Protein science : a publication of the Protein Society
oai:repository.helmholtz-hzi.de:10033/6214762018-09-13T01:34:27Zcom_10033_311308col_10033_559591
Recent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products.
Guttenberger, Nikolaus
Blankenfeldt, Wulf
Breinbauer, Rolf
TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany.
Antibiotics
Anticancer
Biofilm
Biosynthesis
Natural product
Phenazine
Phenazines are natural products which are produced by bacteria or by archaeal Methanosarcina species. The tricyclic ring system enables redox processes, which producing organisms use for oxidation of NADH or for the generation of reactive oxygen species (ROS), giving them advantages over other microorganisms. In this review we summarize the progress in the field since 2005 regarding the isolation of new phenazine natural products, new insights in their biological function, and particularly the now almost completely understood biosynthesis. The review is complemented by a description of new synthetic methods and total syntheses of phenazines.
2018-09-12T09:44:37Z
2018-09-12T09:44:37Z
2018-09-12T09:44:37Z
2017-11-15
Article
1464-3391
28094222
10.1016/j.bmc.2017.01.002
http://hdl.handle.net/10033/621476
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Bioorganic & medicinal chemistry
oai:repository.helmholtz-hzi.de:10033/6214462019-08-30T11:25:41Zcom_10033_311308col_10033_559591
Recent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products.
Guttenberger, Nikolaus
Blankenfeldt, Wulf
Breinbauer, Rolf
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Antibiotics
Anticancer
Biofilm
Biosynthesis
Natural product
Phenazine
Phenazines are natural products which are produced by bacteria or by archaeal Methanosarcina species. The tricyclic ring system enables redox processes, which producing organisms use for oxidation of NADH or for the generation of reactive oxygen species (ROS), giving them advantages over other microorganisms. In this review we summarize the progress in the field since 2005 regarding the isolation of new phenazine natural products, new insights in their biological function, and particularly the now almost completely understood biosynthesis. The review is complemented by a description of new synthetic methods and total syntheses of phenazines.
2018-08-14T10:47:40Z
2018-08-14T10:47:40Z
2018-08-14T10:47:40Z
2017-11-15
Article
1464-3391
28094222
10.1016/j.bmc.2017.01.002
http://hdl.handle.net/10033/621446
en
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Bioorganic & medicinal chemistry
oai:repository.helmholtz-hzi.de:10033/6214512019-08-30T11:28:48Zcom_10033_311308col_10033_559591
Crystal Structures of R-Type Bacteriocin Sheath and Tube Proteins CD1363 and CD1364 From in the Pre-assembled State.
Schwemmlein, Nina
Pippel, Jan
Gazdag, Emerich-Mihai
Blankenfeldt, Wulf
Clostridium difficile
R-type
crystal structure
diffocin
phage tail-like bacteriocins
sheath
tube
iffocins are high-molecular-weight phage tail-like bacteriocins (PTLBs) that some Clostridium difficile strains produce in response to SOS induction. Similar to the related R-type pyocins from Pseudomonas aeruginosa, R-type diffocins act as molecular puncture devices that specifically penetrate the cell envelope of other C. difficile strains to dissipate the membrane potential and kill the attacked bacterium. Thus, R-type diffocins constitute potential therapeutic agents to counter C. difficile-associated infections. PTLBs consist of rigid and contractile protein complexes. They are composed of a baseplate, receptor-binding tail fibers and an inner needle-like tube surrounded by a contractile sheath. In the mature particle, the sheath and tube structure form a complex network comprising up to 200 copies of a sheath and a tube protein each. Here, we report the crystal structures together with small angle X-ray scattering data of the sheath and tube proteins CD1363 (39 kDa) and CD1364 (16 kDa) from C. difficile strain CD630 in a monomeric pre-assembly form at 1.9 and 1.5 Å resolution, respectively. The tube protein CD1364 displays a compact fold and shares highest structural similarity with a tube protein from Bacillus subtilis but is remarkably different from that of the R-type pyocin from P. aeruginosa. The structure of the R-type diffocin sheath protein, on the other hand, is highly conserved. It contains two domains, whereas related members such as bacteriophage tail sheath proteins comprise up to four, indicating that R-type PTLBs may represent the minimal protein required for formation of a complete sheath structure. Comparison of CD1363 and CD1364 with structures of PTLBs and related assemblies suggests that several conformational changes are required to form complete assemblies. In the sheath, rearrangement of the flexible N- and C-terminus enables extensive interactions between the other subunits, whereas for the tube, such contacts are primarily established by mobile α-helices. Together, our results combined with information from structures of homologous assemblies allow constructing a preliminary model of the sheath and tube assembly from R-type diffocin.
2018-08-26T12:07:03Z
2018-08-26T12:07:03Z
2018-08-26T12:07:03Z
2018-01-01
Article
1664-302X
30127773
10.3389/fmicb.2018.01750
http://hdl.handle.net/10033/621451
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6088184/
PMC6088184
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Frontiers in microbiology
oai:repository.helmholtz-hzi.de:10033/6214922019-08-30T11:29:45Zcom_10033_620626com_10033_311308col_10033_620627col_10033_559591
TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.
Zmora, Pawel
Hoffmann, Markus
Kollmus, Heike
Moldenhauer, Anna-Sophie
Danov, Olga
Braun, Armin
Winkler, Michael
Schughart, Klaus
Pöhlmann, Stefan
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
TMPRSS11A
influenza virus
protease
protease inhibitor
virology
virus entry
The influenza virus hemagglutinin (HA) facilitates viral entry into target cells. Cleavage of HA by host cell proteases is essential for viral infectivity, and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease (TTSP) TMPRSS2 has been identified as an HA activator in cell culture and in the infected host. However, it is less clear whether TMPRSS2-related enzymes can also activate HA for spread in target cells. Moreover, the activity of cellular serine protease inhibitors against HA-activating TTSPs is poorly understood. Here, we show that TMPRSS11A, another member of the TTSP family, cleaves and activates the influenza A virus (FLUAV) HA and the Middle East respiratory syndrome coronavirus spike protein (MERS-S). Moreover, we demonstrate that TMPRSS11A is expressed in murine tracheal epithelium, which is a target of FLUAV infection, and in human trachea, suggesting that the protease could support FLUAV spread in patients. Finally, we show that HA activation by the TMPRSS11A-related enzymes human airway tryptase and DESC1, but not TMPRSS11A itself, is blocked by the cellular serine protease inhibitor hepatocyte growth factor activator inhibitor type-1 (HAI-1). Our results suggest that TMPRSS11A could promote FLUAV spread in target cells and that HA-activating TTSPs exhibit differential sensitivity to blockade by cellular serine protease inhibitors.
2018-09-24T12:46:44Z
2018-09-24T12:46:44Z
2018-09-24T12:46:44Z
2018-09-07
Article
1083-351X
29976755
10.1074/jbc.RA118.001273
http://hdl.handle.net/10033/621492
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
The Journal of biological chemistry
oai:repository.helmholtz-hzi.de:10033/6215092019-08-30T11:31:22Zcom_10033_620626com_10033_311308col_10033_620627col_10033_559591
Exchange of amino acids in the H1-haemagglutinin to H3 residues is required for efficient influenza A virus replication and pathology in Tmprss2 knock-out mice.
Lambertz, Ruth L O
Pippel, Jan
Gerhauser, Ingo
Kollmus, Heike
Anhlan, Darisuren
Hrincius, Eike R
Krausze, Joern
Kühn, Nora
Schughart, Klaus
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
hemagglutinin
host protease
influenza A virus
The haemagglutinin (HA) of H1N1 and H3N2 influenza A virus (IAV) subtypes has to be activated by host proteases. Previous
studies showed that H1N1 virus cannot replicate efficiently in Tmprss2/ knock-out mice whereas H3N2 viruses are able to
replicate to the same levels in Tmprss2/ as in wild type (WT) mice. Here, we investigated the sequence requirements for
the HA molecule that allow IAV to replicate efficiently in the absence of TMPRSS2. We showed that replacement of the H3 for
the H1-loop sequence (amino acids 320 to 329, at the C-terminus of HA1) was not sufficient for equal levels of virus
replication or severe pathology in Tmprss2/ knock-out mice compared to WT mice. However, exchange of a distant amino
acid from H1 to H3 sequence (E31D) in addition to the HA-loop substitution resulted in virus replication in Tmprss2/ knockout mice that was comparable to WT mice. The higher virus replication and lung damage was associated with increased
epithelial damage and higher mortality. Our results provide further evidence and insights into host proteases as a promising
target for therapeutic intervention of IAV infections.
2018-10-08T13:36:18Z
2018-10-08T13:36:18Z
2018-10-08T13:36:18Z
2018-09-01
Article
1465-2099
30084768
10.1099/jgv.0.001128
http://hdl.handle.net/10033/621509
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
The Journal of general virology
oai:repository.helmholtz-hzi.de:10033/6215382019-08-30T11:29:11Zcom_10033_311308col_10033_559591
Pseudomonas aeruginosa pyoverdine maturation enzyme PvdP has a noncanonical domain architecture and affords insight into a new subclass of tyrosinases
Poppe, Juliane
Reichelt, Joachim
Blankenfeldt, Wulf
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Pseudomonas aeruginosa (P. aeruginosa)
biosynthesis
copper
enzyme
infectious disease
protein structure
siderophore
streptavidin
tyrosinase
Pyoverdines (PVDs) are important chromophore-containing siderophores of fluorescent pseudomonad bacteria such as the opportunistic human pathogen
2018-11-06T09:49:44Z
2018-11-06T09:49:44Z
2018-11-06T09:49:44Z
2018-09-21
Article
1083-351X
30030378
10.1074/jbc.RA118.002560
http://hdl.handle.net/10033/621538
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
The Journal of biological chemistry
oai:repository.helmholtz-hzi.de:10033/6215572020-08-05T10:54:26Zcom_10033_311308col_10033_559591
Structural insights into antigen recognition of an anti-β-(1,6)-β-(1,3)-D-glucan antibody.
Sung, Kwang Hoon
Josewski, Jörn
Dübel, Stefan
Blankenfeldt, Wulf
Rau, Udo
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Schizophyllan (SCH) is a high molecular weight homopolysaccharide composed of a β-(1,3)-D-glucan main chain with branching β-(1,6)-bound D-glucose residues. It forms triple helices that are highly stable towards heat and extreme pH, which provides SCH with interesting properties for industrial and medical applications. The recombinant anti-SCH antibody JoJ48C11 recognizes SCH and related β-(1,6)-branched β-(1,3)-D-glucans, but details governing its specificity are not known. Here, we fill this gap by determining crystal structures of the antigen binding fragment (Fab) of JoJ48C11 in the apo form and in complex with the unbranched β-(1,3)-D-glucose hexamer laminarihexaose 3.0 and 2.4 Å resolution, respectively. Together with docking studies, this allowed construction of a JoJ48C11/triple-helical SCH complex, leading to the identification of eight amino acid residues of JoJ48C11 (Tyr27
2018-11-13T12:50:41Z
2018-11-13T12:50:41Z
2018-11-13T12:50:41Z
2018-09-12
Article
2045-2322
30209318
10.1038/s41598-018-31961-x
http://hdl.handle.net/10033/621557
560034
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Scientific reports
oai:repository.helmholtz-hzi.de:10033/6215752019-08-30T11:29:42Zcom_10033_338554com_10033_311308col_10033_559591col_10033_338544col_10033_620574
Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.
Schaks, Matthias
Singh, Shashi P
Kage, Frieda
Thomason, Peter
Klünemann, Thomas
Steffen, Anika
Blankenfeldt, Wulf
Stradal, Theresia E
Insall, Robert H
Rottner, Klemens
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Arp2/3 complex
CRISPR/CAS9
Rho-GTPase
filopodium
lamellipodium
migration
protrusion
Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.
2018-11-20T13:29:37Z
2018-11-20T13:29:37Z
2018-11-20T13:29:37Z
2018-10-25
Article
1879-0445
30393033
10.1016/j.cub.2018.10.002
http://hdl.handle.net/10033/621575
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Current biology : CB
oai:repository.helmholtz-hzi.de:10033/6216342019-08-30T11:31:21Zcom_10033_620533com_10033_311308col_10033_620534col_10033_620777col_10033_559591
Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.
Muthukumar, Yazh
Münkemer, Johanna
Mathieu, Daniel
Richter, Christian
Schwalbe, Harald
Steinmetz, Heinrich
Kessler, Wolfgang
Reichelt, Joachim
Beutling, Ulrike
Frank, Ronald
Büssow, Konrad
van den Heuvel, Joop
Brönstrup, Mark
Taylor, Richard E
LASCHAT, SABINE
Sasse, Florenz
The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.
2019-01-07T14:43:20Z
2019-01-07T14:43:20Z
2019-01-07T14:43:20Z
2018-01-01
Article
PLoS One. 2018 Jul 31;13(7):e0201605. doi: 10.1371/journal.pone.0201605 eCollection 2018.
1932-6203
30063768
10.1371/journal.pone.0201605
http://hdl.handle.net/10033/621634
PLOSOne
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
PLOS
PloS one
oai:repository.helmholtz-hzi.de:10033/6216662019-01-23T01:25:10Zcom_10033_311308col_10033_559591
A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine
Friedel, Kristina
Popp, Monika A.
Matern, Julian C. J.
Gazdag, Emerich M.
Thiel, Ilka V.
Volkmann, Gerrit
Blankenfeldt, Wulf
Mootz, Henning D.
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (−1) extein position compared to the parent intein, in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(−1) residue, including D-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N–S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (−1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(−1)Phe and Gly(−1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the extein–intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an extein residue in protein splicing. It sheds new light on the extein–intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins.
2019-01-22T14:59:54Z
2019-01-22T14:59:54Z
2019-01-22T14:59:54Z
2018-10-03
Article
2041-6520
2041-6539
10.1039/C8SC01074A
http://hdl.handle.net/10033/621666
Chemical Science
en
http://xlink.rsc.org/?DOI=C8SC01074A
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Royal Society of Chemistry
10
1
239
251
Chemical Science
oai:repository.helmholtz-hzi.de:10033/6217752019-08-30T11:30:26Zcom_10033_620589com_10033_620652com_10033_311308col_10033_620672col_10033_620777col_10033_620608col_10033_559591
ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.
Büssow, Konrad
Themann, Philipp
Luu, Sabine
Pentrowski, Paul
Harting, Claudia
Majewski, Mira
Vollmer, Veith
Köster, Mario
Grashoff, Martina
Zawatzky, Rainer
van den Heuvel, Joop
Kröger, Andrea
Böldicke, Thomas
HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNβ. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNβ-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.
2019-05-15T07:43:02Z
2019-05-15T07:43:02Z
2019-05-15T07:43:02Z
2019-01-01
Article
PLoS One. 2019 Apr 16;14(4):e0215062. doi: 10.1371/journal.pone.0215062. eCollection 2019.
1932-6203
30990863
10.1371/journal.pone.0215062
http://hdl.handle.net/10033/621775
PLOS ONE
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Plos
PloS one
oai:repository.helmholtz-hzi.de:10033/6218072019-08-30T11:32:37Zcom_10033_311308col_10033_559591
Position 123 of halohydrin dehalogenase HheG plays an important role in stability, activity, and enantioselectivity.
Solarczek, Jennifer
Klünemann, Thomas
Brandt, Felix
Schrepfer, Patrick
Wolter, Mario
Jacob, Christoph R
Blankenfeldt, Wulf
Schallmey, Anett
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral β-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability, which poses a significant drawback for potential applications. In an attempt to thermostabilize HheG by protein engineering, several single mutants at position 123 were identified which displayed up to 14 °C increased apparent melting temperatures and up to three-fold higher activity. Aromatic amino acids at position 123 resulted even in a slightly higher enantioselectivity. Crystal structures of variants T123W and T123G revealed a flexible loop opposite to amino acid 123. In variant T123G, this loop adopted two different positions resulting in an open or partially closed active site. Classical molecular dynamics simulations confirmed a high mobility of this loop. Moreover, in variant T123G this loop adopted a position much closer to residue 123 resulting in denser packing and increased buried surface area. Our results indicate an important role for position 123 in HheG and give first structural and mechanistic insight into the thermostabilizing effect of mutations T123W and T123G.
2019-06-07T14:20:54Z
2019-06-07T14:20:54Z
2019-06-07T14:20:54Z
2019-03-25
Article
Sci Rep. 2019 Mar 25;9(1):5106. doi: 10.1038/s41598-019-41498-2.
2045-2322
30911023
10.1038/s41598-019-41498-2
http://hdl.handle.net/10033/621807
Scientific reports
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Nature publishing group
Scientific reports
oai:repository.helmholtz-hzi.de:10033/6218552019-08-30T11:25:41Zcom_10033_311308col_10033_559591
Crystal Structure of Dihydro-Heme d Dehydrogenase NirN from Pseudomonas aeruginosa Reveals Amino Acid Residues Essential for Catalysis.
Klünemann, Thomas
Preuß, Arne
Adamczack, Julia
Rosa, Luis F M
Harnisch, Falk
Layer, Gunhild
Blankenfeldt, Wulf
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
NirN
denitrification
heme d(1)
tetrapyrrole biosynthesis
x-ray structure
Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called "denitrification" and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd1 nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d1 as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d1. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d1-binding β-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d1 at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417.
2019-07-09T13:40:06Z
2019-07-09T13:40:06Z
2019-07-09T13:40:06Z
2019-06-04
Article
J Mol Biol. 2019 Jun 4. pii: S0022-2836(19)30336-5. doi: 10.1016/j.jmb.2019.05.046.
1089-8638
31173777
10.1016/j.jmb.2019.05.046
http://hdl.handle.net/10033/621855
Journal of Molecular Biology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Elsevier
Journal of molecular biology
oai:repository.helmholtz-hzi.de:10033/6218782019-08-30T11:26:10Zcom_10033_311308col_10033_559591
Crystal structures and protein engineering of three different penicillin G acylases from Gram-positive bacteria with different thermostability.
Mayer, Janine
Pippel, Jan
Günther, Gabriele
Müller, Carolin
Lauermann, Anna
Knuuti, Tobias
Blankenfeldt, Wulf
Jahn, Dieter
Biedendieck, Rebekka
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Bacillus megaterium
Crystal structure
Gram-positive
Penicillin G acylase
Thermostable
Penicillin G acylase (PGA) catalyzes the hydrolysis of penicillin G to 6-aminopenicillanic acid and phenylacetic acid, which provides the precursor for most semisynthetic penicillins. Most applications rely on PGAs from Gram-negative bacteria. Here we describe the first three crystal structures for PGAs from Gram-positive Bacilli and their utilization in protein engineering experiments for the manipulation of their thermostability. PGAs from Bacillus megaterium (BmPGA, Tm = 56.0 °C), Bacillus thermotolerans (BtPGA, Tm = 64.5 °C), and Bacillus sp. FJAT-27231 (FJAT-PGA, Tm = 74.3 °C) were recombinantly produced with B. megaterium, secreted, purified to apparent heterogeneity, and crystallized. Structures with resolutions of 2.20 Å (BmPGA), 2.27 Å (BtPGA), and 1.36 Å (FJAT-PGA) were obtained. They revealed high overall similarity, reflecting the high identity of up to approx. 75%. Notably, the active center displays a deletion of more than ten residues with respect to PGAs from Gram-negatives. This enlarges the substrate binding site and may indicate a different substrate spectrum. Based on the structures, ten single-chain FJAT-PGAs carrying artificial linkers were produced. However, in all cases, complete linker cleavage was observed. While thermostability remained in the wild-type range, the enzymatic activity dropped between 30 and 60%. Furthermore, four hybrid PGAs carrying subunits from two different enzymes were successfully produced. Their thermostabilities mostly lay between the values of the two mother enzymes. For one PGA increased, enzyme activity was observed. Overall, the three novel PGA structures combined with initial protein engineering experiments provide the basis for establishment of new PGA-based biotechnological processes.
2019-07-16T12:12:45Z
2019-07-16T12:12:45Z
2019-07-16T12:12:45Z
2019-06-21
Article
Appl Microbiol Biotechnol. 2019 Jun 21. pii: 10.1007/s00253-019-09977-8. doi: 10.1007/s00253-019-09977-8.
1432-0614
31227867
10.1007/s00253-019-09977-8
http://hdl.handle.net/10033/621878
Applied Microbiology and Biotechnology
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Springer
Applied microbiology and biotechnology
oai:repository.helmholtz-hzi.de:10033/6219212019-08-30T11:24:30Zcom_10033_311308col_10033_559591
Single domain antibodies for the knockdown of cytosolic and nuclear proteins.
Böldicke, Thomas
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
camelid VHHs
cytosolic/nuclear intrabodies
human VH
human VL
intrabodies
scFv fragment
shark vNARs
single domain antibodies
Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant‐ and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell‐penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell‐specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb‐DNA or –protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.
Keywords: intrabodies, single domain antibodies, scFv fragment, cytosolic/nuclear intrabodies, camelid VHHs, shark vNARs, human VH, human VL
2019-08-27T11:02:54Z
2019-08-27T11:02:54Z
2019-08-27T11:02:54Z
2017-01-01
Article
1469-896X
28271570
http://hdl.handle.net/10033/621921
Protein Science
PMC5405437
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405437/
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley-Blackwell
Protein science : a publication of the Protein Society
oai:repository.helmholtz-hzi.de:10033/6219632019-10-01T07:08:45Zcom_10033_311308col_10033_559591col_10033_620561
The N‐terminal peptide of the transglutaminase‐activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross‐linking sites
Juettner, Norbert E.
Schmelz, Stefan
Anderl, Anita
Colin, Felix
Classen, Moritz
Pfeifer, Felicitas
Scrima, Andrea
Fuchsbauer, Hans‐Lothar
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Cell Biology
Biochemistry
Molecular Biology
Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI-family proteins. ENZYMES: Chymotrypsin, EC3.4.21.1; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), EC3.4.24.77; streptogrisin-A (SGPA), EC3.4.21.80; streptogrisin-B (SGPB), EC3.4.21.81; subtilisin BPN', EC3.4.21.62; transglutaminase, EC2.3.2.13; transglutaminase-activating metalloprotease (TAMP), EC3.4.-.-; tri-/tetrapeptidyl aminopeptidase, EC3.4.11.-; trypsin, EC3.4.21.4. DATABASES: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).
2019-09-30T12:19:06Z
2019-09-30T12:19:06Z
2019-09-30T12:19:06Z
2019-08-29
Article
FEBS J. 2019 Aug 17. doi: 10.1111/febs.15044.
1742-464X
1742-4658
10.1111/febs.15044
http://hdl.handle.net/10033/621963
FEBS journal
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley
oai:repository.helmholtz-hzi.de:10033/6220292020-09-29T12:06:33Zcom_10033_311308com_10033_620656col_10033_620657col_10033_559591
Flexible Fragment Growing Boosts Potency of Quorum Sensing Inhibitors against Pseudomonas aeruginosa Virulence.
Zender, Michael
Witzgall, Florian
Kiefer, Alexander Felix
Kirsch, Benjamin
Maurer, Christine K
Kany, Andreas M
Xu, Ningna
Schmelz, Stefan
Börger, Carsten
Blankenfeldt, Wulf
Empting, Martin
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Enthalpic Efficiency
Fragment-based Drug Discovery
Pathoblocker
Pseudomonas aeruginosa
quorum sensing
Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-amino pyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa . We pursue an innovative treatment strategy by interfering with the Pseudomonas Quinolone Signal (PQS) Quorum Sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency.
2019-11-26T10:21:56Z
2019-11-26T10:21:56Z
2019-11-26T10:21:56Z
2019-11-11
Article
ChemMedChem. 2019 Nov 11. doi: 10.1002/cmdc.201900621.
1860-7187
31709767
10.1002/cmdc.201900621
http://hdl.handle.net/10033/622029
ChemMedChem
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley-VCH
ChemMedChem
oai:repository.helmholtz-hzi.de:10033/6220822020-01-15T02:19:41Zcom_10033_311308col_10033_559591
Insights into the Cnx1E catalyzed MPT-AMP hydrolysis.
Hercher, Thomas W
Krausze, Joern
Hoffmeister, Sven
Zwerschke, Dagmar
Lindel, Thomas
Blankenfeldt, Wulf
Mendel, Ralf R
Kruse, Tobias
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Adenylated molybdopterin
Enzyme catalyzed hydrolysis
Molybdenum cofactor
Molybdenum insertase
Molybdenum insertases (Mo-insertases) catalyze the final step of molybdenum cofactor (Moco) biosynthesis, an evolutionary old and highly conserved multi-step pathway. In the first step of the pathway, GTP serves as substrate for the formation of cyclic pyranopterin monophosphate, which is subsequently converted into molybdopterin (MPT) in the second pathway step. In the following synthesis steps, MPT is adenylated yielding MPT-AMP that is subsequently used as substrate for enzyme catalyzed molybdate insertion. Molybdate insertion and MPT-AMP hydrolysis are catalyzed by the Mo-insertase E-domain. Earlier work reported a highly conserved aspartate residue to be essential for Mo-insertase functionality. In this work, we confirmed the mechanistic relevance of this residue for the Arabidopsis thaliana Mo-insertase Cnx1E. We found that the conservative substitution of Cnx1E residue Asp274 by Glu (D274E) leads to an arrest of MPT-AMP hydrolysis and hence to the accumulation of MPT-AMP. We further showed that the MPT-AMP accumulation goes in hand with the accumulation of molybdate. By crystallization and structure determination of the Cnx1E variant D274E, we identified the potential reason for the missing hydrolysis activity in the disorder of the region spanning amino acids 269 to 274. We reasoned that this is caused by the inability of a glutamate in position 274 to coordinate the octahedral Mg2+-water complex in the Cnx1E active site.
2020-01-14T14:56:59Z
2020-01-14T14:56:59Z
2020-01-14T14:56:59Z
2020-01-31
Article
Hercher, Thomas W., et al. "Insights into the Cnx1E catalyzed MPT-AMP hydrolysis." Bioscience Reports (2019): BSR20191806.Hercher, Thomas W., et al. "Insights into the Cnx1E catalyzed MPT-AMP hydrolysis." Bioscience Reports (2019): BSR20191806.
1573-4935
31860061
10.1042/BSR20191806
http://hdl.handle.net/10033/622082
Bioscience Reports
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Portland Press
Bioscience reports
oai:repository.helmholtz-hzi.de:10033/6221142020-02-04T02:37:03Zcom_10033_311308com_10033_620618col_10033_620721col_10033_620619col_10033_559591
The Alkylquinolone Repertoire of Pseudomonas aeruginosa is Linked to Structural Flexibility of the FabH-like 2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS) Biosynthesis Enzyme PqsBC.
Witzgall, Florian
Depke, Tobias
Hoffmann, Michael
Empting, Martin
Brönstrup, Mark
Müller, Rolf
Blankenfeldt, Wulf
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
conformation analysis
enzymes
protein structures
structure-activity relationships
transferases
Pseudomonas aeruginosa is a bacterial pathogen that causes life-threatening infections in immunocompromised patients. It produces a large armory of saturated and mono-unsaturated 2-alkyl-4(1H)-quinolones (AQs) and AQ N-oxides (AQNOs) that serve as signaling molecules to control the production of virulence factors and that are involved in membrane vesicle formation and iron chelation; furthermore, they also have, for example, antibiotic properties. It has been shown that the β-ketoacyl-acyl-carrier protein synthase III (FabH)-like heterodimeric enzyme PqsBC catalyzes the last step in the biosynthesis of the most abundant AQ congener, 2-heptyl-4(1H)-quinolone (HHQ), by condensing octanoyl-coenzyme A (CoA) with 2-aminobenzoylacetate (2-ABA), but the basis for the large number of other AQs/AQNOs produced by P. aeruginosa is not known. Here, we demonstrate that PqsBC uses different medium-chain acyl-CoAs to produce various saturated AQs/AQNOs and that it also biosynthesizes mono-unsaturated congeners. Further, we determined the structures of PqsBC in four different crystal forms at 1.5 to 2.7 Å resolution. Together with a previous report, the data reveal that PqsBC adopts open, intermediate, and closed conformations that alter the shape of the acyl-binding cavity and explain the promiscuity of PqsBC. The different conformations also allow us to propose a model for structural transitions that accompany the catalytic cycle of PqsBC that might have broader implications for other FabH-enzymes, for which such structural transitions have been postulated but have never been observed.
2020-02-03T09:51:38Z
2020-02-03T09:51:38Z
2020-02-03T09:51:38Z
2018-07-16
Article
Chembiochem. 2018 Jul 16;19(14):1531-1544. doi: 10.1002/cbic.201800153. Epub 2018 Jun 22.
1439-7633
29722462
10.1002/cbic.201800153
http://hdl.handle.net/10033/622114
ChemBioChem
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley-VCH
Chembiochem : a European journal of chemical biology
oai:repository.helmholtz-hzi.de:10033/6221272020-02-20T02:04:04Zcom_10033_311308col_10033_559591
Molecular Mechanisms of Vaspin Action - From Adipose Tissue to Skin and Bone, from Blood Vessels to the Brain.
Weiner, Juliane
Zieger, Konstanze
Pippel, Jan
Heiker, John T
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Adiposity
Atherosclerosis
Crystal structure
Exosite
Inflammation
Insulin resistance
Metabolic syndrome
Serine proteases
Serpin
Visceral adipose tissue-derived serine protease inhibitor (vaspin) or SERPINA12 according to the serpin nomenclature was identified together with other genes and gene products that were specifically expressed or overexpressed in the intra-abdominal or visceral adipose tissue (AT) of the Otsuka Long-Evans Tokushima fatty rat. These rats spontaneously develop visceral obesity, insulin resistance, hyperinsulinemia and -glycemia, as well as hypertension and thus represent a well suited animal model of obesity and related metabolic disorders such as type 2 diabetes.The follow-up study reporting the cloning, expression and functional characterization of vaspin suggested the great and promising potential of this molecule to counteract obesity induced insulin resistance and inflammation and has since initiated over 300 publications, clinical and experimental, that have contributed to uncover the multifaceted functions and molecular mechanisms of vaspin action not only in the adipose, but in many different cells, tissues and organs. This review will give an update on mechanistic and structural aspects of vaspin with a focus on its serpin function, the physiology and regulation of vaspin expression, and will summarize the latest on vaspin function in various tissues such as the different adipose tissue depots as well as the vasculature, skin, bone and the brain.
2020-02-11T15:28:19Z
2020-02-11T15:28:19Z
2020-02-11T15:28:19Z
2019-01-01
Article
Adv Exp Med Biol. 2019;1111:159-188. doi: 10.1007/5584_2018_241.
0065-2598
30051323
10.1007/5584_2018_241
http://hdl.handle.net/10033/622127
Advances in Experimental Medicine and Biology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Springer
Advances in experimental medicine and biology
oai:repository.helmholtz-hzi.de:10033/6222682020-05-23T03:32:11Zcom_10033_311308col_10033_620721col_10033_559591
Crystal structure of NirF: insights into its role in heme d biosynthesis.
Klünemann, Thomas
NIMTZ, MANFRED
Jänsch, Lothar
Layer, Gunhild
Blankenfeldt, Wulf
HZI, Helmholtz Zentrum für Infektionsforschung, GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
NirF
X-ray structure
denitrification
heme d1
tetrapyrrole biosynthesis
Certain facultative anaerobes such as the opportunistic human pathogen Pseudomonas aeruginosa can respire on nitrate, a process generally known as denitrification. This enables denitrifying bacteria to survive in anoxic environments and contributes, for example, to the formation of biofilm, hence increasing difficulties in eradicating P. aeruginosa infections. A central step in denitrification is the reduction of nitrite to nitric oxide by nitrite reductase NirS, an enzyme that requires the unique cofactor heme d1 . While heme d1 biosynthesis is mostly understood, the role of the essential periplasmatic protein NirF in this pathway remains unclear. Here, we have determined crystal structures of NirF and its complex with dihydroheme d1 , the last intermediate of heme d1 biosynthesis. We found that NirF forms a bottom-to-bottom β-propeller homodimer and confirmed this by multi-angle light and small-angle X-ray scattering. The N termini are adjacent to each other and project away from the core structure, which hints at simultaneous membrane anchoring via both N termini. Further, the complex with dihydroheme d1 allowed us to probe the importance of specific residues in the vicinity of the ligand binding site, revealing residues not required for binding or stability of NirF but essential for denitrification in experiments with complemented mutants of a ΔnirF strain of P. aeruginosa. Together, these data suggest that NirF possesses a yet unknown enzymatic activity and is not simply a binding protein of heme d1 derivatives. DATABASE: Structural data are available in PDB database under the accession numbers 6TV2 and 6TV9.
2020-05-22T15:04:33Z
2020-05-22T15:04:33Z
2020-05-22T15:04:33Z
2020-04-07
Article
Klünemann T, Nimtz M, Jänsch L, Layer G, Blankenfeldt W. Crystal structure of NirF: insights into its role in heme d1 biosynthesis [published online ahead of print, 2020 Apr 7]. FEBS J. 2020;10.1111/febs.15323. doi:10.1111/febs.15323
32255259
10.1111/febs.15323
http://hdl.handle.net/10033/622268
1742-4658
The FEBS journal
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley Online Open
The FEBS journal
England
oai:repository.helmholtz-hzi.de:10033/6222832020-06-06T01:29:20Zcom_10033_311308col_10033_559591
Biocatalysts from Biosynthetic Pathways: Enabling Stereoselective, Enzymatic Cycloether Formation on a Gram Scale
Hollmann, Tim
Berkhan, Gesche
Wagner, Lisa
Sung, Kwang Hoon
Kolb, Simon
Geise, Hendrik
Hahn, Frank
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Biosynthetic pathways of natural products contain many enzymes that contribute to the rapid assembly of molecular complexity. Enzymes that form complex structural elements with multiple stereocenters, like chiral saturated oxygen heterocycles (CSOH), are of particular interest for a synthetic application, as their use promises to significantly simplify access to these elements. Here, the biocatalytic characterization of AmbDH3, an enzyme that catalyzes intramolecular oxa-Michael addition (IMOMA) is reported. This reaction essentially gives access to various types of CSOH with adjacent stereocenters, but it is not yet part of the repertoire of preparative biocatalysis. An in-depth study on the synthetic utility of AmbDH3 was performed, which made extensive use of complex synthetic precursor surrogates. The enzyme exhibited stability and broad substrate tolerance in in vitro experiments, which was in agreement with the results of molecular modeling. Its selectivity profile enabled kinetic resolution of chiral tetrahydropyrans (THPs) under control of up to four stereocenters. A systematic optimization of the reaction conditions enabled gram-scale conversions yielding preparative amounts of chiral THP. The synthetic utility of AmbDH3 was finally demonstrated by its successful application in the key step of a chemoenzymatic total synthesis to the THP-containing phenylheptanoid (−)-centrolobine. These results highlight the synthetic potential of AmbDH3 and related IMOMA cyclases as a biocatalytic alternative that further develops the available chemical-synthetic IMOMA methodology.
2020-06-05T12:52:06Z
2020-06-05T12:52:06Z
2020-06-05T12:52:06Z
2020-03-30
Article
2155-5435
10.1021/acscatal.9b05071
http://hdl.handle.net/10033/622283
2155-5435
ACS Catalysis
10.1021/acscatal.9b05071
en
http://pubs.acs.org/page/policy/authorchoice_termsofuse.html
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
American Chemical Society (ACS)
10
9
4973
4982
ACS Catalysis
oai:repository.helmholtz-hzi.de:10033/6223302020-07-02T01:31:05Zcom_10033_311308col_10033_559591
The crystal structure of the heme d biosynthesis-associated small c-type cytochrome NirC reveals mixed oligomeric states in crystallo.
Klünemann, Thomas
Henke, Steffi
Blankenfeldt, Wulf
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
3D domain swapping
NirC
Pseudomonas aeruginosa
cytochrome c
heme d1 biosynthesis
Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC.
2020-07-01T12:22:16Z
2020-07-01T12:22:16Z
2020-07-01T12:22:16Z
2020-03-25
Article
Acta Crystallogr D Struct Biol. 2020;76(Pt 4):375-384. doi:10.1107/S2059798320003101.
32254062
10.1107/S2059798320003101
http://hdl.handle.net/10033/622330
2059-7983
Acta crystallographica. Section D, Structural biology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
: International Union of Crystallography
76
Pt 4
375
384
Acta crystallographica. Section D, Structural biology
United States
oai:repository.helmholtz-hzi.de:10033/6223312020-07-02T01:31:11Zcom_10033_311308col_10033_559591
Structure of heme d-free cd nitrite reductase NirS.
Klünemann, Thomas
Blankenfeldt, Wulf
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
NirS
cd1 nitrite reductase
heme d1
A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by the cd1 nitrite reductase NirS in, for example, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d1 as an essential part of its active site. Although NirS has been well studied mechanistically and structurally, the focus of previous studies has been on the active heme d1-bound form. The heme d1-free form of NirS reported here, which represents a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, the movement of a loop around Trp498 seems to be related to a widening of the propeller, allowing easier access to the heme d1-binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed.
2020-07-01T12:37:30Z
2020-07-01T12:37:30Z
2020-07-01T12:37:30Z
2020-05-29
Article
Acta Crystallogr F Struct Biol Commun. 2020;76(Pt 6):250-256. doi:10.1107/S2053230X20006676.
32510465
10.1107/S2053230X20006676
http://hdl.handle.net/10033/622331
2053-230X
Acta crystallographica. Section F, Structural biology communications
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
International Union of Crystallography
76
Pt 6
250
256
Acta crystallographica. Section F, Structural biology communications
United States
oai:repository.helmholtz-hzi.de:10033/6225042020-10-10T01:32:35Zcom_10033_311308col_10033_559591
Gastrointestinal stress as innate defence against microbial attack.
Panwar, H
Rokana, N
Aparna, S V
Kaur, J
Singh, A
Singh, J
Singh, K S
Chaudhary, V
Puniya, A K
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
GI tract
commensal
gut immunity
host-microbe interactions
immunology
pathogens
probiotics
A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF-2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid-encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by-product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better-controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF-2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.
2020-10-09T08:30:24Z
2020-10-09T08:30:24Z
2020-10-09T08:30:24Z
2020-08-31
Review
Biotechnol Bioeng. 2020 Sep 3. doi: 10.1002/bit.27553. Epub ahead of print.
32869386
10.1111/jam.14836
http://hdl.handle.net/10033/622504
1365-2672
Journal of applied microbiology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley
Journal of applied microbiology
England
oai:repository.helmholtz-hzi.de:10033/6225422020-11-04T04:37:26Zcom_10033_311308com_10033_620618com_10033_620656col_10033_620657col_10033_620619col_10033_559591
Protein-Templated Hit Identification through an Ugi Four-Component Reaction.
Mancini, Federica
Unver, M Yagiz
Elgaher, Walid A M
Jumde, Varsha R
Alhayek, Alaa
Lukat, Peer
Herrmann, Jennifer
Witte, Martin D
Köck, Matthias
Blankenfeldt, Wulf
Müller, Rolf
Hirsch, Anna K H
HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.;HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Ugi reaction
drug discovery
kinetic target-guided synthesis
protein-protein interaction inhibitors
protein-templated reactions
2020-10-27T14:46:49Z
2020-10-27T14:46:49Z
2020-10-27T14:46:49Z
2020-05-19
Article
Chemistry. 2020 May 19. doi: 10.1002/chem.202002250.
32428268
10.1002/chem.202002250
http://hdl.handle.net/10033/622542
1521-3765
Chemistry (Weinheim an der Bergstrasse, Germany)
en
info:eu-repo/grantAgreement/EC/H2020/: 757913
http://creativecommons.org/licenses/by-nc-sa/4.0/
openAccess
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley-VCH
Chemistry (Weinheim an der Bergstrasse, Germany)
Germany
oai:repository.helmholtz-hzi.de:10033/6225992020-11-20T01:45:43Zcom_10033_311308col_10033_559591
Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca.
Rodriguez Buitrago, Jhon Alexander
Klünemann, Thomas
Blankenfeldt, Wulf
Schallmey, Anett
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Thermobifida fusca
cytochrome P450
ferredoxin reductase
he ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three-component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe-4S]-cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Å resolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD-binding domain, an NAD-binding domain and a C-terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD- and NAD-binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD-binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD-binding site of FdR9 contains a modified Rossmann-fold motif, GxSxxS, instead of the classical GxGxxG motif.
2020-11-19T14:24:48Z
2020-11-19T14:24:48Z
2020-11-19T14:24:48Z
2020-07-28
Article
Acta Crystallogr F Struct Biol Commun. 2020 Aug 1;76(Pt 8):334-340. doi: 10.1107/S2053230X2000922X. Epub 2020 Jul 28.
32744244
10.1107/S2053230X2000922X
http://hdl.handle.net/10033/622599
2053-230X
Acta crystallographica. Section F, Structural biology communications
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley & Sons
76
Pt 8
334
340
Acta crystallographica. Section F, Structural biology communications
United States
oai:repository.helmholtz-hzi.de:10033/6226142020-12-04T03:37:53Zcom_10033_311308col_10033_559591
CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering.
Bracco, Paula
Wijma, Hein J
Nicolai, Bastian
Rodriguez Buitrago, Jhon Alexander
Klünemann, Thomas
Vila, Agustina
Schrepfer, Patrick
Blankenfeldt, Wulf
Janssen, Dick B
Schallmey, Anett
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
biocatalysis
cytochrome P450 monooxygenase
protein engineering
regioselectivity
steroid hydroxylation
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16a-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that water entrance to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5a-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.
2020-11-26T09:36:36Z
2020-11-26T09:36:36Z
2020-11-26T09:36:36Z
2020-11-04
Article
Chembiochem. 2020 Nov 4. doi: 10.1002/cbic.202000735. Epub ahead of print.
33145893
10.1002/cbic.202000735
http://hdl.handle.net/10033/622614
1439-7633
Chembiochem : a European journal of chemical biology
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Wiley
Chembiochem : a European journal of chemical biology
Germany
oai:repository.helmholtz-hzi.de:10033/6226882021-01-29T11:12:46Zcom_10033_311308col_10033_559591col_10033_620561
The N-terminal peptide of the transglutaminase-activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross-linking sites.
Juettner, Norbert E
Schmelz, Stefan
Anderl, Anita
Colin, Felix
Classen, Moritz
Pfeifer, Felicitas
Scrima, Andrea
Fuchsbauer, Hans-Lothar
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Streptomyces mobaraensis
crystal structure
metalloprotease inhibitor
serine protease inhibitor
transglutaminase
Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI-family proteins. ENZYMES: Chymotrypsin, EC3.4.21.1; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), EC3.4.24.77; streptogrisin-A (SGPA), EC3.4.21.80; streptogrisin-B (SGPB), EC3.4.21.81; subtilisin BPN', EC3.4.21.62; transglutaminase, EC2.3.2.13; transglutaminase-activating metalloprotease (TAMP), EC3.4.-.-; tri-/tetrapeptidyl aminopeptidase, EC3.4.11.-; trypsin, EC3.4.21.4. DATABASES: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).
2021-01-19T11:41:53Z
2021-01-19T11:41:53Z
2021-01-19T11:41:53Z
2019-08-29
Article
FEBS J. 2020 Feb;287(4):708-720. doi: 10.1111/febs.15044. Epub 2019 Aug 29.
31420998
10.1111/febs.15044
http://hdl.handle.net/10033/622688
1742-4658
The FEBS journal
en
http://creativecommons.org/licenses/by-NC-ND/4.0
Attribution 4.0 International
FEBS Press
287
4
708
720
The FEBS journal
England
oai:repository.helmholtz-hzi.de:10033/6227422021-02-16T03:17:51Zcom_10033_311624com_10033_6839com_10033_311308com_10033_338554col_10033_311625col_10033_620721col_10033_559591col_10033_338544
Crystal structure of bacterial cytotoxic necrotizing factor CNFy reveals molecular building blocks for intoxication.
Chaoprasid, Paweena
Lukat, Peer
Mühlen, Sabrina
Heidler, Thomas
Gazdag, Emerich-Mihai
Dong, Shuangshuang
Bi, Wenjie
Rüter, Christian
Kirchenwitz, Marco
Steffen, Anika
Jänsch, Lothar
Stradal, Theresia E B
Dersch, Petra
Blankenfeldt, Wulf
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Yersinia
AB-toxin
ADP-ribosyl transferase
CNF
DUF4765
Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNFY . CNFY consists of five domains (D1-D5), and by integrating structural and functional data, we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) and for a fused β-lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNFY , leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad-specificity protein delivery tool.
2021-02-15T14:54:01Z
2021-02-15T14:54:01Z
2021-02-15T14:54:01Z
2021-01-07
Article
EMBO J. 2021 Jan 7:e105202. doi: 10.15252/embj.2020105202. Epub ahead of print.
33410511
10.15252/embj.2020105202
http://hdl.handle.net/10033/622742
1460-2075
The EMBO journal
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Springer
e105202
The EMBO journal
England
oai:repository.helmholtz-hzi.de:10033/6227472021-02-18T04:37:43Zcom_10033_311308col_10033_559591
Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.
Scheithauer, Lina
Thiem, Stefanie
Schmelz, Stefan
Dellmann, Ansgar
Büssow, Konrad
Brouwer, René M H J
Ünal, Can M
Blankenfeldt, Wulf
Steinert, Michael
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Legionella pneumophila
alveolar septal thickness
collagen IV
crystal structure
human lung tissue explants
zinc metalloprotease ProA
ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.
2021-02-17T14:13:17Z
2021-02-17T14:13:17Z
2021-02-17T14:13:17Z
2021-01-24
Article
Cell Microbiol. 2021 Jan 24:e13313. doi: 10.1111/cmi.13313. Epub ahead of print.
33491325
10.1111/cmi.13313
http://hdl.handle.net/10033/622747
1462-5822
Cellular microbiology
en
http://creativecommons.org/licenses/by-nc-nd/4.0/
Attribution-NonCommercial-NoDerivatives 4.0 International
Wiley
e13313
Cellular microbiology
England
oai:repository.helmholtz-hzi.de:10033/6227682021-03-04T03:35:37Zcom_10033_311308col_10033_559591
Immunotherapy with antibodies: Tumor development, immune defense and therapeutic antibodies
Böldicke, Thomas
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
CD8 - and CD4 -T‑cells + +
Driver genes
Neoantigens
Phases of tumor development
Tumor associated antigens
Tumor development is based on mutations of genes involved in cell growth (e.g. transcription factors, growth receptors or intracellular signal molecules) or in suppressor genes (e.g. p53). During tumor growth cell clones are selected, which contain driver genes, leading to uncontrolled growth of these cell clones. During all phases of tumor development (immunosurveillance, equilibrium phase, escape of the tumor from the immune system) the interaction between the immune system and the tumor cells and the development of a chronic inflammation in the tumor microenvironment play a crucial role. The aim of cancer immunotherapy is to activate the immune system. A promising immunotherapy is based on antibodies that activate immune cells, inhibit tumor growth or lead to destruction of tumor cells. Applied are recombinant IgG antibodies or genetically engineered antibody fragments against tumor-associated antigens (TAA’s). They are applied singularly or in combination with chemo- or radiotherapy. Promising are checkpoint antibodies, which abrogate blocking of cytotoxic CD8+ T cells and CD4+ T cells by tumor cells and/or dendritic cells. Other successfully applied antibodies are bispecific antibodies (recognize T‑cell and tumor cell), chimeric antigen receptors (CARs) for T cell therapy, immunocytokines (cytokines fused to antibodies) and immunotoxins (toxins fused to antibodies). In addition intracellular antibodies successfully tested in xenograft tumor mouse models have promising therapeutic potential.
2021-03-03T16:03:31Z
2021-03-03T16:03:31Z
2021-03-03T16:03:31Z
2021-01-01
Article
Böldicke, T. Immuntherapie mit Antikörpern. Onkologe (2021). https://doi.org/10.1007.
09478965
10.1007/s00761-020-00895-3
http://hdl.handle.net/10033/622768
14330415
Onkologe
2-s2.0-85100213784
SCOPUS_ID:85100213784
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
American Association for Cancer Research (AACR)
Onkologe
oai:repository.helmholtz-hzi.de:10033/6228302021-04-20T02:44:46Zcom_10033_620533com_10033_620613com_10033_311308com_10033_620656col_10033_620534col_10033_620614col_10033_620657col_10033_559591
A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms
Schütz, Christian
Ho, Duy‐Khiet
Hamed, Mostafa Mohamed
Abdelsamie, Ahmed Saad
Röhrig, Teresa
Herr, Christian
Kany, Andreas Martin
Rox, Katharina
Schmelz, Stefan
Siebenbürger, Lorenz
Wirth, Marius
Börger, Carsten
Yahiaoui, Samir
Bals, Robert
Scrima, Andrea
Blankenfeldt, Wulf
Horstmann, Justus Constantin
Christmann, Rebekka
Murgia, Xabier
Koch, Marcus
Berwanger, Aylin
Loretz, Brigitta
Hirsch, Anna Katharina Herta
Hartmann, Rolf Wolfgang
Lehr, Claus‐Michael
Empting, Martin
HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.
General Engineering
General Physics and Astronomy
General Materials Science
Medicine (miscellaneous)
General Chemical Engineering
Biochemistry, Genetics and Molecular Biology (miscellaneous)
Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry‐driven hit‐to‐lead optimization and in‐depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter‐gene with IC50 values as low as 200 and 11 × 10−9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI‐tobramycin (Tob) combination against PA biofilms using a tailor‐made squalene‐derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32‐fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker‐mediated therapy against PA infections opening up avenues for preclinical development.
2021-04-19T10:50:45Z
2021-04-19T10:50:45Z
2021-04-19T10:50:45Z
2021-03-18
Article
Adv. Sci. 2021, 2004369. https://doi.org/10.1002/advs.202004369.
2198-3844
10.1002/advs.202004369
http://hdl.handle.net/10033/622830
2198-3844
Advanced Science
10.1002/advs.202004369
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Wiley and Sons Inc.
2004369
Advanced Science
oai:repository.helmholtz-hzi.de:10033/6228712021-05-18T01:59:32Zcom_10033_311308col_10033_559591
Antimicrobial resistance dynamics and the one-health strategy: a review
Singh, Kumar Siddharth
Anand, Santosh
Dholpuria, Sunny
Sharma, Jitendra Kumar
Blankenfeldt, Wulf
Shouche, Yogesh
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Environmental Chemistry
Antimicrobial resistance is a global threat that kills at least 75,000 people every year worldwide and causes extended hospital stays. In the coming 10 years, antimicrobial resistance is projected to have huge health and economic burden on countries, and the scarcity of available antibiotics further worsens the situation. Antimicrobial resistance results mainly from indiscriminate antibiotic usage in humans, animals and agriculture, and from the rapid emergence and dissemination of resistant pathogens. This issue is challenging for antibiotic stewardship, strict regulations on antibiotics usage, large-scale surveillance and responsible public behavior. This demands international cooperation and integrated efforts under the ‘one-health’ strategy. Here, we review antimicrobial resistance and the one-health strategy. We discuss the historical issue of using antibiotics. We highlight the effectiveness of hygiene in livestock rearing, careful antibiotic usage and large-scale surveillance of animals, humans and environment domains. We present strategies for mitigation of antimicrobial resistance, exemplified by the successful ban of triclosan which induced a significant decline of resistant pathogens. We emphasize the benefits of the global antibiotic resistance partnership and of the one-health participation of stakeholders from public, healthcare professionals and government to mitigate antimicrobial resistance.
2021-05-17T08:59:53Z
2021-05-17T08:59:53Z
2021-05-17T08:59:53Z
2021-04-15
Review
Environ Chem Lett (2021). https://doi.org/10.1007/s10311-021-01238-3.
1610-3653
10.1007/s10311-021-01238-3
http://hdl.handle.net/10033/622871
1610-3661
Environmental Chemistry Letters
1238
en
https://www.springer.com/tdm
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Springer Science and Business Media LLC
Environmental Chemistry Letters
oai:repository.helmholtz-hzi.de:10033/6229142021-07-01T01:43:54Zcom_10033_311308col_10033_620777col_10033_559591
Reproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells.
Schubert, Maren
Nimtz, Manfred
Bertoglio, Federico
Schmelz, Stefan
Lukat, Peer
van den Heuvel, Joop
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Expression vector
High five
Insect cells
TGE
Transient gene expression
he expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.
2021-06-30T15:31:55Z
2021-06-30T15:31:55Z
2021-06-30T15:31:55Z
2021-05-21
Article
Methods Mol Biol. 2021;2305:129-140. doi: 10.1007/978-1-0716-1406-8_6.
33950387
10.1007/978-1-0716-1406-8_6
http://hdl.handle.net/10033/622914
1940-6029
Methods in molecular biology (Clifton, N.J.)
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Springer
2305
129
140
Methods in molecular biology (Clifton, N.J.)
United States
oai:repository.helmholtz-hzi.de:10033/6229462021-07-21T01:57:50Zcom_10033_311308col_10033_559591
NAD(H)-mediated tetramerization controls the activity of phospholipase PlaB.
Diwo, Maurice
Michel, Wiebke
Aurass, Philipp
Kuhle-Keindorf, Katja
Pippel, Jan
Krausze, Joern
Wamp, Sabrina
Lang, Christina
Blankenfeldt, Wulf
Flieger, Antje
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
activation
deoligomerization
membrane association
nicotinamide dinucleotide
phospholipase
The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal α/β-hydrolase domain expanded by two noncanonical two-stranded β-sheets, β-6/β-7 and β-9/β-10. The C-terminal domain reveals a fold displaying a bilobed β-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. Δβ-9/β-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.
2021-07-20T12:47:27Z
2021-07-20T12:47:27Z
2021-07-20T12:47:27Z
2021-06-01
Article
Proc Natl Acad Sci U S A. 2021 Jun 8;118(23):e2017046118. doi: 10.1073/pnas.2017046118.
34074754
10.1073/pnas.2017046118
http://hdl.handle.net/10033/622946
1091-6490
Proceedings of the National Academy of Sciences of the United States of America
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
National Academy of Sciences
118
23
Proceedings of the National Academy of Sciences of the United States of America
United States
oai:repository.helmholtz-hzi.de:10033/6232132022-06-14T01:56:17Zcom_10033_311308col_10033_559591
Structural basis of ergothioneine biosynthesis.
Stampfli, Anja R
Blankenfeldt, Wulf
Seebeck, Florian P
Ergothioneine is a sulfur-containing histidine derivative
synthesized by many bacteria and most fungi but it also finds its
way into human tissue by way of specific absorption from the diet.
The precise role of ergothioneine is not yet known but there is
growing evidence that it plays a role as an antioxidant protecting
human cells from oxidative stress and pathogenic bacteria from
host defenses. In this review we highlight recent advances in
understanding the structural basis of ergothioneine biosynthesis.
In addition to unusual carbon–sulfur bond forming enzymology this
research has revealed that ergothioneine biosynthesis has
emerged at least three times by independent molecular evolution.
2022-06-13T10:12:06Z
2022-06-13T10:12:06Z
2022-06-13T10:12:06Z
2020-05-11
Article
32408082
10.1016/j.sbi.2020.04.002
http://hdl.handle.net/10033/623213
1879-033X
Current opinion in structural biology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
65
1
8
Current opinion in structural biology
England