title: Solution structure and NH exchange studies of the MutT pyrophosphohydrolase complexed with Mg(2+) and 8-oxo-dGMP, a tightly bound product.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Massiah
authors.forename: Vibhor
authors.initials: V
authors.lastname: Saraswat
authors.forename: Hugo F
authors.initials: HF
authors.lastname: Azurmendi
authors.forename: Albert S
authors.initials: AS
authors.lastname: Mildvan
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D003848:Deoxyguanine Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*; D006860:Hydrogen Bonding; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity
chemical_list: D003848:Deoxyguanine Nucleotides; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012996:Solutions; D006157:Guanosine Monophosphate; C039543:8-oxoguanosine 2'-phosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; C052211:mutT protein, E coli; D008274:Magnesium