Crystal structures of an NH2-terminal fragment of T4 DNA polymerase and its complexes with single-stranded DNA and with divalent metal ions
Crystal structures of an NH2-terminal fragment of T4 DNA polymerase and its complexes with single-stranded DNA and with divalent metal ions
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DOI：
10.1021/bi960178r
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发表时间：
1996-06-25
期刊：
影响因子：
2.9
通讯作者：
Steitz, TA
中科院分区：
文献类型：
--
作者：
Wang, J;Yu, P;Steitz, TA
我们报告了以2.2 Angstrom分辨率精制的T4 DNA聚合酶（蛋白质N388）的NH2末端388二级残留片段的晶体结构。该片段既包含3'-5'核酸酶的活性位点，也包含自体mRNA结合位点的一部分（J. D. Karam，个人通信）。载脂蛋白N388和底物P（dt）（3）之间的复合物的结构已以2.5 angstrom的分辨率进行完善，以晶体学R-FACTOR为18.7％。两个二价金属离子辅因子Zn（II）和Mn（II）位于蛋白质N388的晶体中，这些晶体已浸泡在含有Zn（II），Mn（PI）或两者的溶液中。蛋白质N388的3'-5'外切核酸酶结构域与Klenow片段中的相应区域非常相似，尽管序列身份很少。用作催化所需的两个金属离子的配体的四个羧酸盐残基的侧链位于两种蛋白质的几何等效位置，RMS偏差为0.87 Angstrom。两种蛋白质的3'-5'核酸外切酶的活性位点区域之间有两个主要区别：（i）Klenow片段中Tyr-497的OH OH OH OH OH OH OH OH OH OH OH与活性部位中的cissile磷酸盐相互作用（Tyr-320）在蛋白质N388中距离活性中心的点； （ii）底物3'-末端基础的结合袋的不同残基形式。在蛋白质N388络合物中，相对于相应的碱在p（dt）3复合物中与klenow片段中相应的碱基占据的位置相比，P（dt）（3）的3'末端基础旋转约60度。最后，蛋白质N388的单独结构域（残基1-96）可能参与mRNA结合，从而导致T4 DNA聚合酶的翻译调节（Pavlov＆Karam，1994）。
We report the crystal structure of an NH2-terminal 388-residue fragment of T4 DNA polymerase (protein N388) refined at 2.2 Angstrom resolution. This fragment contains both the 3'-5' exonuclease active site and part of the autologous mRNA binding site (J. D. Karam, personal communication). The structure of a complex between the apoprotein N388 and a substrate, p(dT)(3), has been refined at 2.5 Angstrom resolution to a crystallographic R-factor of 18.7%. Two divalent metal ion cofactors, Zn(II) and Mn(II), have been located in crystals of protein N388 which had been soaked in solutions containing Zn(II), Mn(PI), or both. The structure of the 3'-5' exonuclease domain of protein N388 closely resembles the corresponding region in the Klenow fragment despite minimal sequence identity. The side chains of four carboxylate residues that serve as ligands for the two metal ions required for catalysis are located in geometrically equivalent positions in both proteins with a rms deviation of 0.87 Angstrom. There are two main differences between the 3'-5' exonuclease active site regions of the two proteins: (I) the OH of Tyr-497 in the Klenow fragment interacts with the scissile phosphate in the active site whereas the OH of the equivalent tyrosine (Tyr-320) in protein N388 points away from the active center; (II) different residues form of the binding pocket for the 3'-terminal bases of the substrate. In the protein N388 complex the 3'-terminal base of p(dT)(3) is rotated approximately 60 degrees relative to the position that the corresponding base occupies in the p(dT)3 complex with the Klenow fragment. Finally, a separate domain (residues 1-96) of protein N388 may be involved in mRNA binding that results in translational regulation of T4 DNA polymerase (Pavlov & Karam, 1994).