http://hdl.handle.net/10033/6832 Bereich Strukturbiologie (SB) Sun, 26 May 2013 03:45:42 GMT 2013-05-26T03:45:42Z http://hdl.handle.net/10033/283572 Title: Folding and dimerization kinetics of bone morphogenetic protein-2, a member of the transforming growth factor-β family. Authors: Vallejo, Luis F; Rinas, Ursula Abstract: The kinetics of folding and dimerization of bone morphogenetic protein-2 (BMP-2), a disulfide-connected, homodimeric cystine-knot protein and a member of the transforming growth factor-β superfamily, was analyzed under a variety of different conditions. Refolding and dimerization of BMP-2 were extremely slow under all conditions studied, and could be described by consecutive first-order reactions involving at least one long-lived intermediate. The rate constants vary from ~ 0.2 × 10(-5) to ~ 3.5 × 10(-5) s(-1), and were strongly dependent on temperature, redox conditions, and the presence of stabilizing or destabilizing ions. In particular, the combined impact of ionic strength and redox conditions on the rates indicates that electrostatic interactions control thiol-disulfide exchange reactions on the path from the unfolded and reduced monomers to the disulfide-connected growth factor in a rate-determining way. Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10033/283572 2013-01-01T00:00:00Z http://hdl.handle.net/10033/271992 Title: Myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation. Authors: Myllykoski, Matti; Raasakka, Arne; Han, Huijong; Kursula, Petri Abstract: The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2'-nucleotide products from 2',3'-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2'-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain. Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/10033/271992 2012-01-01T00:00:00Z http://hdl.handle.net/10033/270795 Title: Structural characterization of Spinacia oleracea trypsin inhibitor III (SOTI-III). Authors: Glotzbach, Bernhard; Schmelz, Stefan; Reinwarth, Michael; Christmann, Andreas; Heinz, Dirk W; Kolmar, Harald Abstract: In recent decades, several canonical serine protease inhibitor families have been classified and characterized. In contrast to most trypsin inhibitors, those from garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) do not share sequence similarity and have been proposed to form the new Mirabilis serine protease inhibitor family. These 30-40-amino-acid inhibitors possess a defined disulfide-bridge topology and belong to the cystine-knot miniproteins (knottins). To date, no atomic structure of this inhibitor family has been solved. Here, the first structure of S. oleracea trypsin inhibitor III (SOTI-III), in complex with bovine pancreatic trypsin, is reported. The inhibitor was synthesized by solid-phase peptide synthesis on a multi-milligram scale and was assayed to test its inhibitory activity and binding properties. The structure confirmed the proposed cystine-bridge topology. The structural features of SOTI-III suggest that it belongs to a new canonical serine protease inhibitor family with promising properties for use in protein-engineering and medical applications. Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10033/270795 2013-01-01T00:00:00Z http://hdl.handle.net/10033/250993 Title: Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli. Authors: de Groot, Jens C; Schlüter, Kai; Carius, Yvonne; Quedenau, Claudia; Vingadassalom, Didier; Faix, Jan; Weiss, Stefanie M; Reichelt, Joachim; Standfuss-Gabisch, Christine; Lesser, Cammie F; Leong, John M; Heinz, Dirk W; Büssow, Konrad; Stradal, Theresia E B Abstract: Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell. Wed, 07 Sep 2011 00:00:00 GMT http://hdl.handle.net/10033/250993 2011-09-07T00:00:00Z