Binding of non-natural 3'-nucleotides to ribonuclease A.
2'-Fluoro-2'-deoxyuridine 3'-phosphate (dU(F)MP) and arabinouridine 3'-phosphate (araUMP) have non-natural furanose rings. dU(F)MP and araUMP were prepared by chemical synthesis and found to have three- to sevenfold higher affinity than uridine 3'-phosphate (3'-UMP) or 2'-deoxyuridine 3'-phosphate (dUMP) for ribonuclease A (RNase A). These differences probably arise (in part) from the phosphoryl groups of 3'-UMP, dU(F)MP, and araUMP (pK(a) = 5.9) being more anionic than that of dUMP (pK(a) = 6.3). The three-dimensional structures of the crystalline complexes of RNase A with dUMP, dU(F)MP and araUMP were determined at < 1.7 A resolution by X-ray diffraction analysis. In these three structures, the uracil nucleobases and phosphoryl groups bind to the enzyme in a nearly identical position. Unlike 3'-UMP and dU(F)MP, dUMP and araUMP bind with their furanose rings in the preferred pucker. In the RNase A.araUMP complex, the 2'-hydroxyl group is exposed to the solvent. All four 3'-nucleotides bind more tightly to wild-type RNase A than to its T45G variant, which lacks the residue that interacts most closely with the uracil nucleobase. These findings illuminate in atomic detail the interaction of RNase A and 3'-nucleotides, and indicate that non-natural furanose rings can serve as the basis for more potent inhibitors of catalysis by RNase A.
Jenkins
Cara L
CL
Department of Chemistry, University of Wisconsin-Madison, WI, USA.
Thiyagarajan
Nethaji
N
Sweeney
Rozamond Y
RY
Guy
Michael P
MP
Kelemen
Bradley R
BR
Acharya
K Ravi
KR
Raines
Ronald T
RT
D018360:Crystallography, X-Ray; D008958:Models, Molecular; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization
D009711:Nucleotides