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 catalytic 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 determined 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. Biochemistry, preceding paper in this issue) based on distances from the bound metal ion to dTdA and on intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of dTdA, using energy minimization to relieve small 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 Co2+ on 1/T1 and 1/T2 of the phosphorus and on 1/T1 of four proton resonances of dTdA, and these effects are abolished by the binding of the competitive inhibitor 3',5'-pdTp. From the paramagnetic effects of enzyme-bound Co2+ on 1/T1 of phosphorus and protons, with the use of a correlation time of 1.1 ps based on 1/T1 values at 250 and 600 MHz, five metal-nucleus distances and 11 lower limit metal-nucleus distances have been calculated. The Co2+ to 31P distance of 4.1 +/- 0.9 A agrees with that found on the wild-type enzyme (Weber, D. J., Mullen, G. P., & Mildvan, A. S. (1991) Biochemistry 30, 7425-7437) and indicates at least 18% inner sphere phosphate coordination. Fourteen interproton distances and 109 lower limit interproton distances in dTdA in the ternary D21E-La(3+)-dTdA complex were determined by NOESY spectra at 50-, 100-, and 200-ms mixing times. Both the metal-nucleus and interproton distances were necessary to compute a narrow range of conformations for enzyme-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 conformations for the individual nucleosides. However, significant conformational 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).(ABSTRACT TRUNCATED AT 400 WORDS)
Weber
D J
DJ
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.
Libson
A M
AM
Gittis
A G
AG
Lebowitz
M S
MS
Mildvan
A S
AS
D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010758:Phosphorus / Q000737:chemistry; D011487:Protein Conformation; D011522:Protons; D013210:Staphylococcus / Q000201:enzymology; D013816:Thermodynamics; D013942:Thymine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D017186:Titrimetry
D008670:Metals; D011522:Protons; D013942:Thymine Nucleotides; D010758:Phosphorus; C019111:thymidine 3',5'-diphosphate; D003035:Cobalt; D008345:Manganese; D008836:Micrococcal Nuclease