NMR studies of the magnesium-ATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme

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Abstract
Proton NMR was used to study the interaction of .beta.,.gamma.-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind Mg.epsilon.ATP [Hamada, M., Palmieri, R. H., Russell, G. A., and Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 .mu.M, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 .mu.M]. Time-dependent nuclear Overhauser effects (NOE''s) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE''s at 250 and 500 MHz. The H2'' to H1'' distance so obtained (3.07 .ANG.) was within the range established by X-ray and model-building studies of nucleotides (2.9 .+-. 0.2 .ANG.). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (.chi. = 63 .+-. 12.degree.) and 3''-endo/O1''-endo ribose puckers (.delta. = 96 .+-. 12.degree.). Enzyme- and peptide-bound MgATP molecules exhibited different C4''-C5'' torsional angles (.gamma.) of 170.degree. and 50.degree., respectively. Ten intermolecular NOE''s from protons of the enzyme and four such NOE''s from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of .beta.,.gamma.-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual consistency of interproton and Cr3+ to proton distances obtained in metal-ATP complexes of both the enzyme and the peptide suggests that the conformation of the peptide is very similar to that of residues 1-45 of the enzyme. When this was assumed to be the case and when molecular models and a computer graphics system were used, MgATP could be fit into the X-ray structure of adenylate kinase in a unique manner such that all of the distances determined by NMR were accommodated. The adenine ribose moiety is bound in a hydrophobic pocket consisting of residues Ile-28, Val-29, His-36, Leu-37, and Leu-91, while the Mg2+-triphosphate portion binds near Lys-21 and -27 and Gln-24. In this complex, the .gamma.-phosphoryl group of MgATP is directed toward residues 172-194 which, in the form of a peptide, has previously been shown to bind .epsilon.AMP [Hamada, M., Palmieri, R. A., Russell, G. A., and Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The MgATP-binding site determined by NMR is also consistent with the results of chemical modification and kinetic studies and with structural and sequence homologies among many nucleotide binding proteins.