Conserved residues that modulate protein trans-splicing of Npu DnaE split intein

摘要

pThe first crystal itrans/i-structure of a naturally occurring split intein has been determined for the iNpu/i (iNostoc punctiforme/i PCC73102) DnaE split intein. Guided by this structure, the residues supN/supArgsup50/sup and supC/supSersup35/sup, well conserved in DnaE split inteins, are identified to be critical in the itrans/i-splicing of iNpu/i DnaE split intein. An iin vitro/i splicing assay demonstrates that supN/supArgsup50/sup and supC/supSersup35/sup play synergistic roles in modulating its intein activity. The C-terminal supC/supAsnsup36/sup exhibits two orientations of its side chain and interacts with both supN/supArgsup50/sup and supC/supSersup35/sup through hydrogen bonding. These interactions likely facilitate the cyclization of asparagine in the course of protein splicing. The mutation of either residue reduces intein activity, and correlates with the low activity of the iSsp/i (iCyanobacterium synechocystis/i sp. strain PCC6803) DnaE split intein. On the other hand, supN/supArgsup50/sup also forms a hydrogen bond with the highly conserved F-block supC/supAspsup17/sup, thus influencing the N–S acyl shift during N-terminal cleavage. Sequence alignments show that residues supN/supArgsup50/sup and supC/supSersup35/sup are rather conserved in those split inteins that lack a penultimate histidine residue. The conserved non-catalytic residues of split inteins modulate the efficiency of protein itrans/i-splicing by hydrogen-bond interactions with the catalytic residues at the splice junction./p