Solution Structure and NH Exchange Studies of the MutT Pyrophosphohydrolase Complexed with Mg2+ and 8-Oxo-dGMP, a Tightly Bound Product

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• 作者：Michael A. Massiah ; Vibhor Saraswat ; Hugo F. Azurmendi ; and Albert S. Mildvan
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：September 2, 2003
• 年：2003
• 卷：42
• 期：34
• 页码：10140 - 10154
• 全文大小：2135K
• 年卷期：v.42,no.34(September 2, 2003)
• ISSN：1520-4995

文摘

To learn the structural basis for the unusually tight binding of 8-oxo-nucleotides to the MutTpyrophosphohydrolase of Escherichia coli (129 residues), the solution structure of the MutT-Mg²⁺-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 s/gifchars/phi.gif" BORDER=0 > and s/gifchars/psi.gif" BORDER=0 > values derived from backbone ¹⁵N, Cs/gifchars/alpha.gif" BORDER=0>, Hs/gifchars/alpha.gif" BORDER=0>,and Cs/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> chemical shifts, 20 converged structures were computed with NOE violations s/entities/le.gif">0.25 Å and totalenergies s/entities/le.gif">450 kcal/mol. The pairwise root-mean-square deviations (RMSD) of backbone N, Cs/gifchars/alpha.gif" BORDER=0>, and C'atoms for the secondary structured regions and for all residues of the 20 structures are 0.65 and 0.98 Å,respectively, indicating a well-defined structure. Further refinement using residual dipolar coupling from53 backbone N-H vectors slightly improved the RMSD values to 0.49 and 0.84 Å, respectively. Thesecondary structures, which consisted of two s/gifchars/alpha.gif" BORDER=0>-helices and a five-stranded mixed s/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-sheet, wereindistinguishable from those of free MutT and of MutT in the quaternary MutT-Mg²⁺-(H₂O)-AMPCPP-Mg²⁺ complex. Comparisons of these three tertiary structures showed a narrowing of the hydrophobicnucleotide-binding cleft in the 8-oxo-dGMP complex resulting from a 2.5-4.5 Å movement of helix Iand a 1.5 Å movement of helix II and loop 4 toward the cleft. The binding of 8-oxo-dGMP to MutT-Mg²⁺ buries 71-78% of the surface area of the nucleotide. The 10^3.7-fold weaker binding substrate analogueMg²⁺-AMPCPP induced much smaller changes in tertiary structure, and MutT buried only 57% of thesurface of the AMP moiety of AMPCPP. Formation of the MutT-Mg²⁺-8-oxo-dGMP complex slowedthe backbone NH exchange rates of 45 residues of the enzyme by factors of 10¹-10⁶ as compared withthe MutT-Mg²⁺ and the MutT-Mg²⁺-dGMP complexes, suggesting a more compact structure when 8-oxo-dGMP is bound. The 10^4.6-fold weaker binding of dGMP to MutT-Mg²⁺ (K_D = 1.8 mM) slowed thebackbone exchange rates of only 20 residues and by smaller factors of ~10. Hence, the high affinity ofMutT-Mg²⁺ for 8-oxo-dGMP likely results from widespread ligand-induced conformation changes thatnarrow 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-78may also contribute.