CONTRIBUTION OF RNASE SA COMPLEXES TO PROTEIN-PROTEIN/NUCLEIC ACID RECOGNITION

Urbániková L.. & Ševcík J.

Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21 84251 Bratislava, Slovak Republic

RNase Sa is an endoribonuclease secreted by Streptomyces aureofaciens. It specifically hydrolyzes phosphodiester bonds of single-stranded RNA at the 3'-side of guanosine nucleotides. The structure of RNase Sa was solved by multiple isomorphous replacement and refined at various resolutions, including atomic. The crystal structures of the complexes of RNase Sa with mononucleotide inhibitors, guanosine-3'-monophosphate, guanosine-2'-monophosphate and exo-guanosine-2',3'-cyclophosphorothioate were refined at high resolution. These structural studies contributed to better understanding the mechanism of RNase Sa action.

Barstar, a natural polypeptide inhibitor of ribonuclease from Bacillus amyloliquefaciens (barnase), binds to and fully inhibits RNase Sa. The mechanism of inhibition of both enzymes by barstar is very similar, i. e. by steric blocking of the active site. Dissociation constant of the non-natural complex of RNase Sa with barstar (10^-10M), is four orders higher than that of the barnase-barstar complex. Crystal structure of the complex of RNase Sa with barstar was solved by molecular replacement and refined at 1.7 resolution. Refinement converged with R factor of 16.2 %.

RNase Sa residues which are important in binding and cleaving substrate, except Glu 54, are also involved in forming contacts with polypeptide inhibitor. Structures of the complexes of RNase Sa with inhibitors were analysed in details. There are differences in areas of interfaces, and numbers of intermolecular van der Waals contacts, direct hydrogen bonds and hydrogen bonds mediated through water molecules. The above differences rationalize in general terms the differences in dissociation constants of complexes. This analysis and an attempt to quantify the contribution of the forces which are involved in formation of the complexes shed additional light on understanding the principles of protein/protein and protein/nucleic acid recognition.