Document Detail


X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition.
MedLine Citation:
PMID:  16414069     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.
Authors:
Kerstin B Rohr; Trevor Selwood; Ulf Marquardt; Robert Huber; Norman M Schechter; Wolfram Bode; Manuel E Than
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2005-12-28
Journal Detail:
Title:  Journal of molecular biology     Volume:  357     ISSN:  0022-2836     ISO Abbreviation:  J. Mol. Biol.     Publication Date:  2006 Mar 
Date Detail:
Created Date:  2006-03-13     Completed Date:  2006-04-18     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985088R     Medline TA:  J Mol Biol     Country:  England    
Other Details:
Languages:  eng     Pagination:  195-209     Citation Subset:  IM    
Affiliation:
Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18, 82152 Martinsried, Germany.
Data Bank Information
Bank Name/Acc. No.:
PDB/2F9N;  2F9O;  2F9P
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Binding Sites
Cattle
Crystallography, X-Ray
Cysteine Proteinase Inhibitors / chemistry*,  metabolism
Humans
Isoenzymes / chemistry*,  genetics,  metabolism
Leupeptins / chemistry*,  metabolism
Models, Molecular
Molecular Sequence Data
Mutation
Protein Structure, Quaternary*
Recombinant Proteins / chemistry,  genetics,  metabolism
Sequence Alignment
Serine Endopeptidases / chemistry*,  genetics,  metabolism
Tryptases
Chemical
Reg. No./Substance:
0/Cysteine Proteinase Inhibitors; 0/Isoenzymes; 0/Leupeptins; 0/Recombinant Proteins; 24365-47-7/leupeptin; EC 3.4.21.-/Serine Endopeptidases; EC 3.4.21.59/Tryptases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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