Model for the catalytic domain of the proofreading ε subunit of Escherichia coli DNA polymerase III based on NMR structural data
Model for the catalytic domain of the proofreading ε subunit of Escherichia coli DNA polymerase III based on NMR structural data
复制标题
DOI：
10.1021/bi0114170
复制
发表时间：
2002-01-08
期刊：
影响因子：
2.9
通讯作者：
London, RE
中科院分区：
文献类型：
--
作者：
DeRose, EF;Li, DW;London, RE
DNA聚合酶III全酶（HE）是大肠杆菌的主要复制聚合酶。HE复合物的α亚基为该酶复合物提供3 '-外切核酸水解校正活性。ESTs由两个结构域组成：含有校正核酸外切酶活性的N-末端结构域（残基1-186）和与聚合酶（α）亚基结合所需的C-末端结构域（残基187-243）。对H-2-、C-13-和N-15-标记的N-末端结构域（ε 186）进行多维NMR研究，以分配骨架共振并测量H-N-H-N核奥弗豪泽效应（NOE）。还对含有具有质子化甲基的瓦尔、Leu和Ile残基的三重标记的[U-H-2，C-13，N-15] ε 186进行了NMR研究，这允许指定H-N-CH 3和CH 3-CH 3 NOES。使用化学位移索引和TALOS程序分析C-13（α）、C-13（β）和（CO）-C-13位移，可以鉴定二级结构区域。H-N-H-N NOE提供了关于延伸链组装成β折叠结构的信息，并确认了α螺旋的归属。H-N-CH 3和CH 3-CH 3 NOE的测量证实了β-折叠结构，并有助于α螺旋的定位。由此产生的蛋白质的三维结构的初步表征表明，显着的结构同源性存在与Klenow校对核酸外切酶结构域的活性位点，尽管极其有限的序列同源性。在此类比的基础上，ε 186的分子模拟研究使用Klenow片段和T4 DNA聚合酶的外切核酸酶结构域的晶体结构以及最近确定的E.大肠杆菌核酸外切酶I。构建了多序列比对，初始比对取自先前发表的隐马尔可夫模型和NMR约束。由于几个已发表的结构包括复合的ssDNA，我们也能够将A-C-G三核苷酸掺入ε 186结构中。几乎所有被鉴定为突变子的残基都位于结合DNA的分子部分。其中大多数发挥催化或结构作用。
The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzyme complex. epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). Multidimensional NMR studies of H-2-, C-13-, and N-15-labeled N-terminal domains (epsilon186) were performed to assign the backbone resonances and measure H-N-H-N nuclear Overhauser effects (NOEs). NMR studies were also performed on triple-lableled [U-H-2,C-13,N-15]epsilon186 containing Val, Leu, and Ile residues with protonated methyl groups, which allowed for the assignment of H-N-CH3 and CH3-CH3 NOES. Analysis of the C-13(alpha), C-13(beta), and (CO)-C-13 shifts, using chemical shift indexing and the TALOS program, allowed for the identification of regions of the secondary structure. H-N-H-N NOEs provided information on the assembly of the extended strands into a beta-sheet structure and confirmed the assignment of the alpha helices. Measurement of H-N-CH3 and CH3-CH3 NOEs confirmed the beta-sheet structure and assisted in the positioning of the alpha helices. The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the active site of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology. On the basis of this analogy, molecular modeling studies of epsilon186 were performed using as templates the crystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase and the recently determined structure of the E. coli Exonuclease I. A multiple sequence alignment was constructed, with the initial alignment taken from the previously published hidden Markov model and NMR constraints. Because several of the published structures included complexed ssDNA, we were also able to incorporate an A-C-G trinucleotide into the epsilon186 structure. Nearly all of the residues which have been identified as mutators are located in the portion of the molecule which binds the DNA. with most of these playing either a catalytic or structural role.