1pun
|
Solution Structure of the MutT Pyrophosphohydrolase Complexed with Mg(2+) and 8-oxo-dGMP, a Tightly-bound Product
OverviewOverview
To learn the structural basis for the unusually tight binding of, 8-oxo-nucleotides to the MutT pyrophosphohydrolase of Escherichia coli, (129 residues), the solution structure of the MutT-Mg(2+)-8-oxo-dGMP, product complex (K(D) = 52 nM) was determined by standard 3-D, heteronuclear NMR methods. Using 1746 NOEs (13.5 NOEs/residue) and 186 phi, and psi values derived from backbone (15)N, Calpha, Halpha, and Cbeta, chemical shifts, 20 converged structures were computed with NOE violations, <or=0.25 A and total energies <or=450 kcal/mol. The pairwise, root-mean-square deviations (RMSD) of backbone N, Calpha, and C' atoms for, the secondary structured regions and for all residues of the 20 structures, are 0.65 and 0.98 A, respectively, indicating a well-defined structure., Further refinement using residual dipolar coupling from 53 backbone N-H, vectors slightly improved the RMSD values to 0.49 and 0.84 A, respectively. The secondary structures, which consisted of two, alpha-helices and a five-stranded mixed beta-sheet, were indistinguishable, from those of free MutT and of MutT in the quaternary, MutT-Mg(2+)-(H(2)O)-AMPCPP-Mg(2+) complex. Comparisons of these three, tertiary structures showed a narrowing of the hydrophobic, nucleotide-binding cleft in the 8-oxo-dGMP complex resulting from a, 2.5-4.5 A movement of helix I and a 1.5 A movement of helix II and loop 4, toward the cleft. The binding of 8-oxo-dGMP to MutT-Mg(2+) buries 71-78%, of the surface area of the nucleotide. The 10(3.7)-fold weaker binding, substrate analogue Mg(2+)-AMPCPP induced much smaller changes in tertiary, structure, and MutT buried only 57% of the surface of the AMP moiety of, AMPCPP. Formation of the MutT-Mg(2+)-8-oxo-dGMP complex slowed the, backbone NH exchange rates of 45 residues of the enzyme by factors of, 10(1)-10(6) as compared with the MutT-Mg(2+) and the MutT-Mg(2+)-dGMP, complexes, suggesting a more compact structure when 8-oxo-dGMP is bound., The 10(4.6)-fold weaker binding of dGMP to MutT-Mg(2+) (K(D) = 1.8 mM), slowed the backbone exchange rates of only 20 residues and by smaller, factors of approximately 10. Hence, the high affinity of MutT-Mg(2+) for, 8-oxo-dGMP likely results from widespread ligand-induced conformation, changes that narrow the nucleotide binding site and lower the overall free, energy of the enzyme-product complex. Specific hydrogen bonding of the, purine ring of 8-oxo-dGMP by the side chains of Asn-119 and Arg-78 may, also contribute.
About this StructureAbout this Structure
1PUN is a Single protein structure of sequence from Escherichia coli with MG and 8OG as ligands. Full crystallographic information is available from OCA.
ReferenceReference
Solution structure and NH exchange studies of the MutT pyrophosphohydrolase complexed with Mg(2+) and 8-oxo-dGMP, a tightly bound product., Massiah MA, Saraswat V, Azurmendi HF, Mildvan AS, Biochemistry. 2003 Sep 2;42(34):10140-54. PMID:12939141
Page seeded by OCA on Wed Nov 21 00:08:07 2007