4gig

Publication Abstract from PubMed

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. While 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.

Conserved threonine spring-loads precursor for intein splicing.,Dearden AK, Callahan B, Van Roey P, Li Z, Kumar U, Belfort M, Nayak SK Protein Sci. 2013 Feb 19. doi: 10.1002/pro.2236. PMID:23423655^[1]

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