2.50
Hdl Handle:
http://hdl.handle.net/10033/19774
Title:
Thermodynamically reengineering the listerial invasion complex InlA/E-cadherin.
Authors:
Wollert, Thomas; Heinz, Dirk W; Schubert, Wolf-Dieter
Abstract:
Biological processes essentially all depend on the specific recognition between macromolecules and their interaction partners. Although many such interactions have been characterized both structurally and biophysically, the thermodynamic effects of small atomic changes remain poorly understood. Based on the crystal structure of the bacterial invasion protein internalin (InlA) of Listeria monocytogenes in complex with its human receptor E-cadherin (hEC1), we analyzed the interface to identify single amino acid substitutions in InlA that would potentially improve the overall quality of interaction and hence increase the weak binding affinity of the complex. Dissociation constants of InlA-variant/hEC1 complexes, as well as enthalpy and entropy of binding, were quantified by isothermal titration calorimetry. All single substitutions indeed significantly increase binding affinity. Structural changes were verified crystallographically at < or =2.0-A resolution, allowing thermodynamic characteristics of single substitutions to be rationalized structurally and providing unique insights into atomic contributions to binding enthalpy and entropy. Structural and thermodynamic data of all combinations of individual substitutions result in a thermodynamic network, allowing the source of cooperativity between distant recognition sites to be identified. One such pair of single substitutions improves affinity 5,000-fold. We thus demonstrate that rational reengineering of protein complexes is possible by making use of physically distant hot spots of recognition.
Affiliation:
Molecular Host-Pathogen Interactions, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.
Citation:
Thermodynamically reengineering the listerial invasion complex InlA/E-cadherin. 2007, 104 (35):13960-5 Proc. Natl. Acad. Sci. U.S.A.
Journal:
Proceedings of the National Academy of Sciences of the United States of America
Issue Date:
28-Aug-2007
URI:
http://hdl.handle.net/10033/19774
DOI:
10.1073/pnas.0702199104
PubMed ID:
17715295
Type:
Article
Language:
en
ISSN:
0027-8424
Appears in Collections:
Publications from Division of Molekulare Struktur Biologie (MOSB)

Full metadata record

DC FieldValue Language
dc.contributor.authorWollert, Thomas-
dc.contributor.authorHeinz, Dirk W-
dc.contributor.authorSchubert, Wolf-Dieter-
dc.date.accessioned2008-03-05T10:30:35Z-
dc.date.available2008-03-05T10:30:35Z-
dc.date.issued2007-08-28-
dc.identifier.citationThermodynamically reengineering the listerial invasion complex InlA/E-cadherin. 2007, 104 (35):13960-5 Proc. Natl. Acad. Sci. U.S.A.en
dc.identifier.issn0027-8424-
dc.identifier.pmid17715295-
dc.identifier.doi10.1073/pnas.0702199104-
dc.identifier.urihttp://hdl.handle.net/10033/19774-
dc.description.abstractBiological processes essentially all depend on the specific recognition between macromolecules and their interaction partners. Although many such interactions have been characterized both structurally and biophysically, the thermodynamic effects of small atomic changes remain poorly understood. Based on the crystal structure of the bacterial invasion protein internalin (InlA) of Listeria monocytogenes in complex with its human receptor E-cadherin (hEC1), we analyzed the interface to identify single amino acid substitutions in InlA that would potentially improve the overall quality of interaction and hence increase the weak binding affinity of the complex. Dissociation constants of InlA-variant/hEC1 complexes, as well as enthalpy and entropy of binding, were quantified by isothermal titration calorimetry. All single substitutions indeed significantly increase binding affinity. Structural changes were verified crystallographically at < or =2.0-A resolution, allowing thermodynamic characteristics of single substitutions to be rationalized structurally and providing unique insights into atomic contributions to binding enthalpy and entropy. Structural and thermodynamic data of all combinations of individual substitutions result in a thermodynamic network, allowing the source of cooperativity between distant recognition sites to be identified. One such pair of single substitutions improves affinity 5,000-fold. We thus demonstrate that rational reengineering of protein complexes is possible by making use of physically distant hot spots of recognition.en
dc.language.isoenen
dc.subject.meshAmino Acid Substitutionen
dc.subject.meshAsparagineen
dc.subject.meshBacterial Proteinsen
dc.subject.meshBinding Sitesen
dc.subject.meshCadherinsen
dc.subject.meshCalorimetryen
dc.subject.meshGenetic Engineeringen
dc.subject.meshGlycineen
dc.subject.meshHumansen
dc.subject.meshKineticsen
dc.subject.meshListeria monocytogenesen
dc.subject.meshModels, Molecularen
dc.subject.meshProtein Bindingen
dc.subject.meshProtein Conformationen
dc.subject.meshRecombinant Proteinsen
dc.subject.meshSerineen
dc.subject.meshThermodynamicsen
dc.subject.meshTyrosineen
dc.subject.meshVariation (Genetics)en
dc.titleThermodynamically reengineering the listerial invasion complex InlA/E-cadherin.en
dc.typeArticleen
dc.contributor.departmentMolecular Host-Pathogen Interactions, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.en
dc.identifier.journalProceedings of the National Academy of Sciences of the United States of Americaen

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