The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'-p 
[5'-phospho-2'-deoxyuridine-3'-pyrophosphate (P'-->5') adenosine 3'-phosphate] 
has been determined at 1.7 A resolution. This dinucleotide is the most potent 
low molecular weight inhibitor of RNase A reported to date (K(i) = 27 nM) and is 
also effective against two major nonpancreatic RNases: eosinophil-derived 
neurotoxin and RNase-4; in all cases, tight binding in large part derives from 
the unusual 3',5'-pyrophosphate internucleotide linkage [Russo, N., and Shapiro, 
R. (1999) J. Biol. Chem. 274, 14902-14908]. The design of pdUppA-3'-p was based 
on the crystal structure of RNase A complexed with 5'-diphosphoadenosine 
3'-phosphate (ppA-3'-p) [Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., 
and Acharya, K. R. (1997) Biochemistry 36, 5578-5588]. The adenosine of 
pdUppA-3'-p adopts an atypical syn conformation not observed for standard 
adenosine nucleotides bound to RNase A. This conformation, which allows 
extensive interactions with Asn 67, Gln 69, Asn 71, and His 119, is associated 
with the placement of the 5'-beta-phosphate of the adenylate, rather than 
alpha-phosphate, at the site where substrate phosphodiester bond cleavage 
occurs. The contacts of the deoxyuridine 5'-phosphate portion of pdUppA-3'-p 
appear to be responsible for the 9-fold increased affinity of this compound as 
compared to ppA-3'-p: the uracil base binds to Thr 45 in the same manner as 
previous pyrimidine inhibitors, and the terminal 5'-phosphate is positioned to 
form medium-range Coulombic interactions with Lys 66. The full potential benefit 
of these added interactions is not realized because of compensatory losses of 
hydrogen bonds of Lys 7 and Gln 11 with the terminal 3'-phosphate and the 
adenylate 5'-alpha-phosphate, which were not predicted by modeling. The results 
reported here have important implications for the design of improved inhibitors 
of RNase A and for the development of therapeutic agents to control the 
activities of RNase homologues such as eosinophil-derived neurotoxin and 
angiogenin that have roles in human pathologies.