NMR DOCKING OF A SUBSTRATE INTO THE X-RAY STRUCTURE OF THE ASP-21 -] GLU MUTANT OF STAPHYLOCOCCAL NUCLEASE

To understand the structural basis of the 1500-fold decrease in cataly tic activity of the D21E mutant of staphylococcal nuclease in which an aspartate ligand of the essential Ca2+ has been enlarged to glutamate , the conformation of the enzyme-bound substrate dTdA has been determi ned by NMR methods and has been docked into the X-ray structure of the D21E mutant (Libson, A. M., Gittis, A.G., & Lattman, E. E. Biochemist ry, preceding paper in this issue) based on distances from the bound m etal ion to dTdA and on intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 t o proton resonances of dTdA, using energy minimization to relieve smal l overlaps. Like the wild-type enzyme, the D21E mutant forms binary E- M and E-S and ternary E-M-S complexes with Ca2+, Mn2+, Co2+, and La3+. D21E enhances the paramagnetic effects of Co(2+)on (1)/T-1 and (1)/T- 2 of the phosphorus and on (1)/T-1 of four proton resonances of dTdA, and these effects are abolished by the binding of the competitive inhi bitor 3',5'-pdTp. From the paramagnetic effects of enzyme-bound Co2+ O n (1)/T-1 of phosphorus and protons, with the use of a correlation tim e of 1.1 ps based on (1)/T-1 values at 250 and 600 MHz, five metal-nuc leus distances and 11 lower limit metal-nucleus distances have been ca lculated. The Co2+ to P-31 distance of 4.1 +/- 0.9 Angstrom agrees wit h that found on the wild-type enzyme (Weber, D. J., Mullen, G. P., & M ildvan, A. S. (1991) Biochemistry 30, 7425-7437) and indicates at leas t 18% inner sphere phosphate coordination. Fourteen interproton distan ces and 109 lower limit interproton distances in dTdA in the ternary D 21E-La3+-dTdA complex were determined by NOESY spectra at 50-, 100-, a nd 200-ms mixing times. Both the metal-nucleus and interproton distanc es were necessary to compute a narrow range of conformations for enzym e-bound dTdA. As on the wild-type enzyme, the conformation of dTdA on the D21E mutant is highly extended, with high-anti C-2' endo conformat ions for the individual nucleosides. However, significant conformation al differences are found in the torsional angles chi of dA (Delta chi = 49 +/- 3 degrees), in gamma of dT (Delta gamma = 108 +/- 30 degrees) and in zeta of dT (Delta zeta = 124 +/- 38 degrees). Despite these di fferences, in the NMR-docked ternary complex, the angle of nucleophili c attack, defined by the oxygen of an inner sphere water ligand of Ca2 +, the phosphorus, and the leaving 5'-deoxyadenosyl oxygen of dTdA of 139 +/- 17 degrees on D21E is very similar to that found on the wild-t ype enzyme (131 +/- 16 degrees) and is appropriate for inversion. The reaction coordinate distance from the water oxygen to the attacked pho sphorus of 4.8 +/- 0.5 Angstrom agrees within error with that found on the wild-type enzyme (4.3 +/- 0.5 Angstrom) and is appropriate for an associative displacement at phosphorus. The catalytic residues, Arg-3 5 and Arg-87, interact similarly with the substrate on the D21E and wi ld-type enzymes. Hence, while other factors may contribute, the profou nd decrease in catalytic activity of the D21E mutant may result predom inantly from the unavailability of Glu-43, due to its direct coordinat ion by the metal ion, as found in the X-ray structure of the D21E-Ca(2 +)3',5'-pdTp complex (Libson, A., Gittis, A., & Lattman, E. E. (1993) Biochemistry, preceding paper in this issue). A catalytic role for Glu -43 is proposed in which this residue orients the attacking hydroxyl l igand on Ca2+.