CRYSTAL-STRUCTURES OF RIBONUCLEASE-A COMPLEXES WITH 5'-DIPHOSPHOADENOSINE 3'-PHOSPHATE AND 5'-DIPHOSPHOADENOSINE 2'-PHOSPHATE AT 1.7-ANGSTROM RESOLUTION

High-resolution (1.7 Angstrom) crystal structures have been determined for bovine pancreatic ribonuclease A (RNase A) complexed with 5'-diph osphoadenosine 3'-phosphate (ppA-3'-p) and 5'-diphosphoadenosine 2'-ph osphate (ppA-2'-p), as well as for a native structure refined to 2.0 A ngstrom. These nucleotide phosphates are the two most potent inhibitor s of RNase A reported so far, with K-i values of 240 and 520 nM, respe ctively. The binding modes and conformations of ppA-3'-p and ppA-2'-p were found to differ markedly from those anticipated on the basis of e arlier structures of RNase A complexes. The key difference is that the 5'-beta-phosphate rather than the 5'-alpha-phosphate of each inhibito r occupies the P-1 phosphate binding site. As a consequence, the ribos e moieties of the two nucleotides are shifted by similar to 2 Angstrom compared to the positions of their counterparts in earlier complexes, and the adenine rings are rotated into unusual syn conformations. Thu s, the six-membered and five-membered rings of both adenines are rever sed with respect to the others but nonetheless engage in extensive int eractions with the residues that form the B-2 purine binding site of R Nase A. Despite the close structural similarity of the two inhibitors, the puckers of their furanose rings are different: C2'-endo and C3'-e ndo, respectively. Moreover, their 5'-alpha-phosphates and 3'(2')-mono phosphates interact with largely different sets of RNase residues. The results of this crystallographic study emphasize the difficulties inh erent in qualitative modeling of protein-inhibitor interactions and th e compelling reasons for high-resolution structural studies in which q uantitative design of improved inhibitors was enabled. The structures presented here provide a promising starting point for the rational des ign of tight-binding RNase inhibitors, which may be used as therapeuti c agents in restraining the ribonucleolytic activities of RNase homolo gues such as angiogenin, eosinophil-derived neurotoxin, and eosinophil cationic protein.