THE STRUCTURES OF RNASE-A COMPLEXED WITH 3'-CMP AND D(CPA) - ACTIVE-SITE CONFORMATION AND CONSERVED WATER-MOLECULES

The interactions of RNase A with cytidine 3'-monophosphate (3'-CMP) an d deoxycytidyl-3:5'-deoxyadenosine (d(CpA)) were analyzed by X-ray cry stallography. The 3'-CMP complex and the native structure were determi ned from trigonal crystals, and the d(CpA) complex from monoclinic cry stals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein-inhibitor conta cts are interpreted in terms of the catalytic mechanism. The general b ase His 12 interacts with the 2' oxygen, as does the electrostatic cat alyst Lys 41. The general acid His 119 has 2 conformations (A and B) i n the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3'-CMP complex. From the present st ructures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex p ermits a detailed analysis of the downstream binding site, which inclu des His 119 and Asn 71. The comparison of the present RNase A structur es with an inhibitor complex of RNase T1 shows that there are importan t similarities in the active sites of these 2 enzymes, despite the abs ence of any sequence homology. The water molecules were analyzed in or der to identify conserved water sites. Seventeen water sites were foun d to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the bin ding of the N-terminal helix to the rest of the protein and in the sta bilization of the active site.