Toward rational design of ribonuclease inhibitors: High-resolution crystalstructure of a ribonuclease a complex with a potent 3 ',5 '-pyrophosphate-linked dinucleotide inhibitory
Dd. Leonidas et al., Toward rational design of ribonuclease inhibitors: High-resolution crystalstructure of a ribonuclease a complex with a potent 3 ',5 '-pyrophosphate-linked dinucleotide inhibitory, BIOCHEM, 38(32), 1999, pp. 10287-10297
The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'
-p [5'-phospho-2'-deoxyuridine-3'-pyrophosphate (P'-->5') adenosine 3'-phos
phate] has been determined at 1.7 Angstrom 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 ail cases, tight binding in
large part derives from the unusual 3',5'-pyrophosphate internucleotide li
nkage [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 com
plexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) [Leonidas, D. D.,
Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemist
ry 36, 5578-5588]. The adenosine of pdUppA-3'-p adopts an atypical syn conf
ormation not observed for standard adenosine nucleotides bound to RNase A.
This conformation, which allows extensive interactions with Asn 67, Gin 69,
Asn 71, and His 119, is associated with the placement of the 5'-beta-phosp
hate of the adenylate, rather than alpha-phosphate, at the site where subst
rate 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 ba
se binds to Thr 45 in the same manner as previous pyrimidine inhibitors, an
d the terminal 5'-phosphate is positioned to form medium-range Coulombic in
teractions with Lys 66. The full potential benefit of these added interacti
ons 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-pho
sphate, which were not predicted by modeling. The results reported here hav
e important implications for the design of improved inhibitors of RNase A a
nd for the development of therapeutic agents to control the activities of R
Nase homologues such as eosinophil-derived neurotoxin and angiogenin that h
ave roles in human pathologies.