In the X‐ray structure of the staphylococcal nuclease–Ca2+ −3′,5′‐pdTp complex, the conformation of the inhibitor 3′,5′‐pdTp is distroteed Lys‐70* and Lys‐71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5 : 183‐201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme‐bound 3′,5′‐pdTp in the staphylococcal nuclease–metal–pdTp Complex determined by NMR methods was docked into the X‐ray structure of the enzyme [Weber, D. J., Serpersu, E. H., Gittis, A. G., Lattman, E. E., Mildvan, A. S. (preceding paper)]. In the NMR‐docked structure, the 5′‐phophate of 3′,5′‐pdTp overlaps with that in the X‐ray Structure. However the 3′‐phosphate accepts a hydrogen bond from Lys‐49 (2.89Å) rather than from Lys‐84 (8.63 Å), and N3 of thymine donates a hydrogen bond to the OH of Tyr‐115 (3.16 Å) which does not occur in the X‐ray structure (5.28 Å). These interactions have been tested by binding studies of 3′,5′‐pdTp, Ca2+, and Mn2+ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two‐dimensional NMR spectroscopy, but bound Ca2+ 9.1‐ to 9.9‐fold more weakly than the wild‐type enzyme. While thye K84A mutation did not significantly weaken 3′,5′‐pdTp binding to the enzyme (1.5 ± 0.7 fold), the K49A mutation weakened 3′,5′‐pdTp binding to the enzyme by the factor of 4.4 ± 1.8‐fold. Similarly, the Y115A mutation weakened 3′,5′‐pdTp binding to the enzyme 3.6 ± 1.6‐fold. Comparable weakening effects of these mutations were found on the binding of Ca2+‐3′,5′‐pdTp. These results are more readily explained by the NMR‐docked structure of staphylococcal nuclease‐metal‐3′,5′‐pdTp than by the X‐ray structure. © 1993 Wiley‐Liss, Inc.
All Science Journal Classification (ASJC) codes
- Structural Biology
- Molecular Biology