NMR DOCKING OF THE COMPETITIVE INHIBITOR THYMIDINE 3',5'-DIPHOSPHATE INTO THE X-RAY STRUCTURE OF STAPHYLOCOCCAL NUCLEASE

In the X-ray structure of the ternary staphylococcal nuclease-Ca2+-3', 5'-pdTp complex, the conformation of the bound inhibitor 3',5'-pdTp is distorted by Lys-70 and Lys-71* from an adjacent molecule of the enz yme in the crystal lattice (Loll, P. J. and Lattman, E. E. Proteins 5: 183-201,1989; Serpersu, E. H., Hibler, D. W., Gerlt, J. A., and Mildva n, A. S. Biochemistry 28:1539-1548, 1989). Since this interaction does not occur in solution, the NMR docking procedure has been used to cor rect this problem. Based on 8 Co2+-nucleus distances measured by param agnetic effects on T1, and 9 measured and 45 lower limit interproton d istances determined by 1D and 2D NOE studies of the ternary Ca2+ compl ex, the conformation of enzyme-bound 3',5'-pdTp is high-anti (chi = 58 +/- 10-degrees) with a C2' endo/O1' endo sugar pucker (delta = 143 +/ - 2-degrees), (-) synclinal about the C3'-O3' bond (epsilon = 273 +/- 4-degrees), trans, gauche about the C4'-C5' bond (gamma = 301 +/- 29-d egrees) and either (-) or (+) clinal about the C5'-O5' bond (beta = 92 +/- 8-degrees or 274 +/- 3-degrees). The structure of 3',5'-pdTp in t he crystalline complex differs due to rotations about the C4'-C5' bond (gamma = 186 +/- 12-degrees, gauche, trans) and the C5'-O5' bond [bet a = 136 +/- 10-degrees, (+) anticlinal]. The undistorted conformation of enzyme-bound metal-3',5'-pdTp determined by NMR was docked into the X-ray structure of the enzyme, using 19 intermolecular NOEs from ring proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonance s of the inhibitor. van der Waals overlaps were then removed by energy minimization. Subsequent molecular dynamics and energy minimization p roduced no significant changes, indicating the structure to be in a gl obal rather than in a local minimum. While the metal-coordinated 5'-ph osphate of the NMR-docked structure of 3',5'-pdTp over-laps with that in the X-ray structure, and similarly receives bifunctional hydrogen b onds from both Arg-35 and Arg-87, the thymine, deoxyribose, and 3'-pho sphate are significantly displaced from their positions in the X-ray s tructure, with the 3'-phosphate receiving hydrogen bonds from Lys-49 r ather than from Lys-84 and Tyr-85. The repositioned thymine ring permi ts hydrogen bonding to the phenolic hydroxyl of Tyr-115. These new int eractions, found in the NMR docked structure, are supported by reduced affinities for 3',5'-pdTp by appropriate mutants of staphylococcal nu clease (Chuang, W.-J., Weber, D.J., Gittis, A.G., and Mildvan, A.S. (1 993) accompanying paper, this issue). An inner sphere, rather than a s econd sphere water ligand of the metal, is optimally positioned to don ate a hydrogen bond to Glu-43 and to attack the coordinated 5'-phospha te with inversion. It is concluded that the NMR docking procedure can be used to correct structural artifacts created by lattice contacts in crystals, when they occur at or near ligand binding sites, such as th e active sites of enzymes. (C) 1993 Wiley-Liss, Inc.