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–Ca 2+ −3′,5′‐pdTp complex, the conformation of the bound inhibitor 3′,5′‐pdTp is distorted by Lys‐70* Abbreviations used: 3′,5′‐pdTp, thymidine 3′,5′‐di‐phosphate; Tris‐HCl, tris‐(hydroxymethyl)aminomethane hydrochloride; NOE, nuclear Overhauser effect; EDTA, ethylene‐diaminetetraacetic acid; TES, N ‐[tris(hydroxymethyl)‐methyl]‐2‐aminoethane sulfonic acid; Lys‐70*, 71*, lysine residues from a neighboring molecule of staphylococcal nuclease in the crystal lattice. and Lys‐71* from an adjacent molecule of the enzyme 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 Mildvan, A.S. Biochemistry 28:1539–1548, 1989). Since this interaction does not occur in solution, the NMR docking procedure has been used to correct this problem. Based on 8 Co 2+ ‐nucleus distances measured by paramagnetic effects on T 1 , and 9 measured and 45 lower limit interproton distances determined by 1D and 2D NOE studies of the ternary Ca 2+ complex, the conformation of enzyme‐bound 3′,5′‐pdTp is high‐anti (χ = 58 = 10°) with a C2′ endo/O1′ endo sugar pucker (δ = 143 ± 2°), (−) synclinal about the C3′‐O3′ bond (ε = 273 ± 4°), trans, gauche about the C4′–C5′ bond (γ = 301 ± 29°) and either (−) or (+) clinal about the C5′–O5′ bond (β = 92 ± 8° or 274 ± 3°). The structure of 3′,5′‐pdTp in the crystalline complex differs due to rotations about the C4′–C5′ bond (γ = 186 ± 12°, gauche, trans) and the C5′–O5′ bond [β = 136 + 10°, (+) 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 resonances of the inhibitor. van der Waals overlaps were then removed by energy minimization. Subsequent molecular dynamics and energy minimization produced no significant changes, indicating the structure to be in a global rather than in a local minimum. While the metal‐coordinated 5′‐phosphate of the NMR‐docked structure of 3′,5′‐pdTp overlaps with that in the X‐ray structure, and similarly receives bifunctional hydrogen bonds from both Arg‐35 and Arg‐87, the thymine, deoxyribose, and 3′‐phosphate are significantly displaced from their positions in the X‐ray structure, with the 3′‐phosphate receiving hydrogen bonds from Lys‐49 rather than from Lys‐84 and Tyr‐85. The repositioned thymine ring permits hydrogen bonding to the phenolic hydroxyl of Tyr‐115. These new interactions, found in the NMR docked structure, are supported by reduced affinities for 3′,5′‐pdTp by appropriate mutants of staphylococcal nuclease (Chuang, W.‐J., Weber, D.J., Gittis, A.G., and Mildvan, A.S. (1993) accompanying paper, this issue). An inner sphere, rather than a second sphere water ligand of the metal, is optimally positioned to donate a hydrogen bond to Glu‐43 and to attack the coordinated 5′‐phosphate 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 the active sites of enzymes. © 1993 Wiley‐Liss, Inc.