Crystallographic studies on a cold adapted subtilase and proteins involved in mRNA processing

Abstract

The crystal structure of a subtilisin-like serine proteinase from the psychrotrophic marine bacterium, Vibrio sp. PA-44, was solved by means of molecular replacement and refined at 1.84 Å resolution. This is the first structure of a cold adapted subtilase to be determined and its elucidation facilitates examination of the molecular principles underlying temperature adaptation of enzymes. The cold adapted Vibrio-proteinase was compared to known three-dimensional structures of homologous enzymes of meso-and thermophilic origin, proteinase K and thermitase, to which it has high structural resemblance. The main structural features standing out as plausible determinants of the different temperature adaptation of the enzymes involve the character of their exposed and buried surface areas. The hydrophobic effect is found to play a significant role for the structural stability of the meso-and thermophile enzymes, whereas the cold enzyme exposes more of its apolar surface. In addition, the cold adapted Vibrio-proteinase is distinguished from the more stable enzymes by its strong anionic character arising from the high occurrence of uncompensated negatively charged residues on its surface.Interestingly, both the cold and thermophile proteinases differ from the mesophile enzyme in having more extensive hydrogen-and ion pair interactions in their structures, supporting suggestions of the dual role of electrostatic interactions in adaptation of enzymes to both high and low temperatures. The Vibrio-proteinase has three calcium ions associated with its structure, one of which is in a calcium-binding site not described in other subtilases.A 61 kDa protein component of the human spliceosome is indispensable for the assembly of the [U4/U6•U5] triple snRNP. The gene encoding the 61 kDa spliceosomal protein was overexpressed in E. coli and purified for crystallisation trials. No crystals were obtained. Evidence of incomplete folding and heterogeneity due to unspecific binding of E. coli nucleic acids emerged in the purification process.A 42 kDa protein component of the Trypanosoma brucei RNA editosome has endo-and exonuclease activity and is suggested to mediate protein-protein and/or protein-RNA interactions. Attempts were made to crystallise the 42 kDa protein with and without non-hydrolysable double stranded nucleic acid ligands but without success.Protein preparation for crystallisation trials of p61 .