期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 316, 期 4, 页码 919-929出版社
ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
DOI: 10.1006/jmbi.2001.5357
关键词
protein-splicing; VMA1-derived endonuclease; intein; thiazolidine intermediate; crystal structure
Protein splicing excises an internal intein segment from a protein precursor precisely, and concomitantly ligates flanking N and C-extein polypeptides at the respective sides of the precursor. Here, a series of precursor recombinants bearing 11 N-extein and ten C-extein residues is prepared for the intein of the Saccharomyces cerevisiae VMAI-derived homing endonuclease referred to as VIDE and as PI-SceI. The recombinant with replacements of C284S, H362N, N737S, and C738S is chosen as a splice-able precursor model and is then subjected to a 2.1 Angstrom resolution crystallographic analysis. The crystal structure shows that the introduced extein polypeptides are located in the vicinity of the splicing site, and that each of their peptide bonds is in the trans conformation. The S284 O-gamma atom located at a distance of 3.1 Angstrom from the G283 C atom in the N-terminal junction suggests that a nucleophilic attack of the C284 S-gamma atom on the G283 C atom forms a tetrahedral intermediate containing a five-membered thiazolidine ring. The tetrahedral intermediate is supposedly resolved into a thioester acyl group upon the cleavage of the linkage between the G283 C and C284 N atoms, and this thioester acyl formation completes the initial steps of N --> S acyl shift at the junction between the N-extein and intein. The S738 O-gamma atom in the C-terminal junction is placed in close proximity to the S284 O-gamma atom at a distance of 3.6 Angstrom, and is well suited for another nucleophilic attack on the resultant thioester acyl group that is then subjected to the transesterification in the next step. The reaction steps proposed for the acyl shift are driven entirely by protonation and deprotonation, in which proton ingress and egress is balanced within the splicing site. (C) 2002 Elsevier Science Ltd.
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