Crystal structure of a splicing precursor of VMA1-derived endonuclease

Department Applied Biochemistry, Tokai University
â—‹Akihiro Sakai Ryuta Mizutani

Protein splicing is a post-translational process in which an internal segment (intein) excises itself out of a precursor with concomitant ligation of the two flanking peptides (extein). Crystallographic analyses have been performed for recombinants of the VMA1-derived endonuclease (VDE) bearing N- and C-extein residues. The crystal structure of the X10SSS recombinant (with mutations C284S / H362N / N737S / C738S) showed that the extein peptides were connected to the intein via trans peptide bonds, while X10SNS (C284S / H362N / C738S) underwent splicing in the crystal lattice. These results suggest that N737 is essential for the protein splicing reaction.
Expression systems of VDE precursors bearing mutation N737S were prepared by cloning mutant genes derived from pTYB1 carrying a VDE allele into the NdeI-BamHI site of the expression vector pET-17b. The VDE precursor with single mutation (N737S) or double mutation (N737S / C738A) was purified by anion-exchange and gel-filtration chromatography. In the SDS-PAGE analysis, each purified protein gave a single 53-kDa band corresponding to the splicing precursor. This band was resistant to reducing agents, indicating that the single mutation N737S inhibits thioester formation in the initial N-S acyl shift step of the protein splicing. The N737S protein was crystallized in the presence of metal ions. Diffraction data to 3 A resolution were collected by a laboratory diffractometer. The initial model obtained by the molecular replacement method using the X10SSS-VDE structure as a search molecule was subjected to the crystallographic refinement. Electron density maps of the splicing sites indicate that the existence of the extein peptides connected to the intein.