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

摘要

The first crystal trans-structure of a naturally occurring split intein has been determined for the Npu (Nostoc punctiforme PCC73102) DnaE split intein. Guided by this structure, the residues (N)Arg(50) and (C)Ser(35), well conserved in DnaE split inteins, are identified to be critical in the trans-splicing of Npu DnaE split intein. An in vitro splicing assay demonstrates that (N)Arg(50) and cSer35 play synergistic roles in modulating its intein activity. The C-terminal (c)Asn(36) exhibits two orientations of its side chain and interacts with both (N)Arg(50) and cSer35 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 Ssp (Cyanobacterium synechocystis sp. strain PCC6803) DnaE split intein. On the other hand, (N)Arg(50) also forms a hydrogen bond with the highly conserved F-block cAse, thus influencing the N-S acyl shift during N-terminal cleavage. Sequence alignments show that residues (N)Arg(5) and cSer35 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 trans-splicing by hydrogen-bond interactions with the catalytic residues at the splice junction