Document Detail


A conserved threonine spring-loads precursor for intein splicing.
MedLine Citation:
PMID:  23423655     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Protein splicing is an autocatalytic process where an "intein" self-cleaves from a precursor and ligates the flanking N- and C-"extein" polypeptides. Inteins occur in all domains of life and have myriad uses in biotechnology. Although the reaction steps of protein splicing are known, mechanistic details remain incomplete, particularly the initial peptide rearrangement at the N-terminal extein/intein junction. Recently, we proposed that this transformation, an N-S acyl shift, is accelerated by a localized conformational strain, between the intein's catalytic cysteine (Cys1) and the neighboring glycine (Gly-1) in the N-extein. That proposal was based on the crystal structure of a catalytically competent trapped precursor. Here, we define the structural origins and mechanistic relevance of the conformational strain using a combination of quantum mechanical simulations, mutational analysis, and X-ray crystallography. Our results implicate a conserved, but largely unstudied, threonine residue of the Ssp DnaE intein (Thr69) as the mediator of conformational strain through hydrogen bonding. Further, the strain imposed by this residue is shown to position the splice junction in a manner that enhances the rate of the N-S acyl shift substantially. Taken together, our results not only provide fundamental understanding of the control of the first step of protein splicing but also have important implications in various biotechnological applications that require precursor manipulation.
Authors:
Albert K Dearden; Brian Callahan; Patrick Van Roey; Zhong Li; Utsav Kumar; Marlene Belfort; Saroj K Nayak
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2013-03-26
Journal Detail:
Title:  Protein science : a publication of the Protein Society     Volume:  22     ISSN:  1469-896X     ISO Abbreviation:  Protein Sci.     Publication Date:  2013 May 
Date Detail:
Created Date:  2013-04-26     Completed Date:  2013-11-06     Revised Date:  2014-05-07    
Medline Journal Info:
Nlm Unique ID:  9211750     Medline TA:  Protein Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  557-63     Citation Subset:  IM    
Copyright Information:
Copyright © 2013 The Protein Society.
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MeSH Terms
Descriptor/Qualifier:
Crystallography, X-Ray
DNA Polymerase III / chemistry,  genetics*
Escherichia coli / chemistry,  genetics*
Inteins*
Models, Molecular
Point Mutation
Protein Conformation
Protein Splicing*
Grant Support
ID/Acronym/Agency:
GM44844/GM/NIGMS NIH HHS; R01 GM044844/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
EC 2.7.7.-/DNA Polymerase III; EC 2.7.7.-/DNA polymerase III, alpha subunit
Comments/Corrections

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