Crystal structure of a hybrid between ribonuclease A and bovine seminal ribonuclease - the basic surface, at 2.0 angstrom resolution

variant of bovine pancreatic ribonuclease A has been prepared with seven am ino acid substitutions (Q55K, N62K, A64T, Y76K, S80RI E111G, N113K). These substitutions recreate in RNase A the basic surface found in bovine seminal RNase, a homologue of pancreatic RNase that diverged some 35 million years ago. Substitution of a portion of this basic surface (positions 55, 62, 64 , 111 and 113) enhances the immunosuppressive activity of the RNase variant , activity found in native seminal RNase. while substitution of another por tion (positions 76 and 80) attenuates the activity. Further, introduction o f Gly at position 111 has been shown to increase the catalytic activity of RNase against double-stranded RNA. The variant and the wild-type (recombina nt) protein were crystallized and their structures determined to a resoluti on of 2.0 Angstrom. Each of the mutated amino acids is seen in the electron density map. The main change observed in the mutant structure compared wit h the wild-type is the region encompassing residues 16-22, where the struct ure is more disordered. This loop is the region where the polypeptide chain of RNase A is cleaved by subtilisin to form RNase S, and undergoes conform ational change to allow residues 1-20 of the RNase to swap between subunits in the covalent seminal RNase dimer.