Massiah Michael A, Saraswat Vibhor, Azurmendi Hugo F, Mildvan Albert S
Department of Biological Chemistry, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Biochemistry. 2003 Sep 2;42(34):10140-54. doi: 10.1021/bi030105p.
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.
为了解8-氧代核苷酸与大肠杆菌MutT焦磷酸水解酶（129个残基）异常紧密结合的结构基础，采用标准的三维异核核磁共振方法测定了MutT-Mg(2+)-8-氧代-dGMP产物复合物（K(D)=52 nM）的溶液结构。利用1746个NOE（13.5个NOE/残基）以及由主链（15）N、Cα、Hα和Cβ化学位移得出的186个φ和ψ值，计算出20个收敛结构，其NOE违反情况≤0.25 Å，总能量≤450 kcal/mol。这20个结构的二级结构区域以及所有残基的主链N、Cα和C'原子的成对均方根偏差（RMSD）分别为0.65 Å和0.98 Å，表明结构明确。使用来自53个主链N-H向量的剩余偶极耦合进行进一步优化后，RMSD值分别略微提高到0.49 Å和0.84 Å。二级结构由两个α螺旋和一个五链混合β折叠组成，与游离MutT以及四级MutT-Mg(2+)-(H(2)O)-AMPCPP-Mg(2+)复合物中的MutT的二级结构无法区分。对这三种三级结构的比较表明，在8-氧代-dGMP复合物中疏水核苷酸结合裂隙变窄，这是由于螺旋I移动2.5 - 4.5 Å以及螺旋II和环4向裂隙移动1.5 Å所致。8-氧代-dGMP与MutT-Mg(2+)的结合掩埋了核苷酸71 - 78%的表面积。结合能力弱10(3.7)倍的底物类似物Mg(2+)-AMPCPP诱导的三级结构变化要小得多，MutT仅掩埋了AMPCPP的AMP部分表面的57%。与MutT-Mg(2+)和MutT-Mg(2+)-dGMP复合物相比，MutT-Mg(2+)-8-氧代-dGMP复合物的形成使该酶45个残基的主链NH交换速率减慢了10(1)-10(6)倍，这表明8-氧代-dGMP结合时结构更紧凑。dGMP与MutT-Mg(2+)的结合能力弱10(4.6)倍（K(D)=1.8 mM），仅使20个残基的主链交换速率减慢，且减慢因子约为10。因此，MutT-Mg(2+)对8-氧代-dGMP的高亲和力可能源于广泛的配体诱导构象变化，这些变化使核苷酸结合位点变窄并降低了酶-产物复合物的整体自由能。8-氧代-dGMP嘌呤环与Asn-119和Arg-78侧链的特异性氢键作用也可能有贡献。
