Crystal structures of Escherichia coli RecA in complex with MgADP and MnAMP-PNP.
Crystal structures of Escherichia coli RecA in complex with MgADP and MnAMP-PNP.
复制标题
大肠杆菌 RecA 与 MgADP 和 MnAMP-PNP 复合物的晶体结构。
DOI：
10.1021/bi048165y
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Bell,CharlesE
中科院分区：
文献类型：
--
作者：
Xing,Xu;Bell,CharlesE
RecA催化同源重组的DNA配对和链交换步骤，这是修复双链DNA断裂的重要机制。RecA与DNA的结合受腺苷核苷酸调节。ATP增加RecA对DNA的亲和力，而ADP降低亲和力。以前，E. coliRecA及其与ADP的复合物的分辨率分别为2.3和3.0 μ m，但RecA-ADP复合物的模型不包括镁离子或侧链。在这里，我们已经确定了RecA与MgADP和MnAMP−PNP（ATP的不可水解类似物）复合物的晶体结构，分辨率分别为1.9和2.1 nm。这两种晶体在相同的条件下生长，并且具有右旋螺旋形式的RecA，螺距为1.82 π。晶体结构显示了RecA与核苷酸辅因子的详细相互作用，包括金属离子和AMP−PNP的γ磷酸盐。与ADP和AMP−PNP结合的RecA的结构之间几乎没有构象差异，与未复合的RecA的区别仅在于核苷酸结合时P环残基66−73的轻微开放。为了解释MnAMP−PNP复合物结构的功能意义，使用辅助蛋白酶测定来比较不同核苷酸促进RecA活性延伸构象的能力。ATPγS和ADP− AlF 4促进了一种强有力的辅蛋白酶活性，而ADP和AMP−PNP根本不能激活RecA。我们得出结论，RecA−MnAMP−PNP复合物的晶体结构代表了RecA蛋白的预异构化状态，该状态存在于ATP结合之后但在构象转变为活性状态之前。
RecA catalyzes the DNA pairing and strand-exchange steps of homologous recombination, an important mechanism for repair of double-stranded DNA breaks. The binding of RecA to DNA is modulated by adenosine nucleotides. ATP increases the affinity of RecA for DNA, while ADP decreases the affinity. Previously, the crystal structures ofE. coliRecA and its complex with ADP have been determined to resolutions of 2.3 and 3.0 Å, respectively, but the model for the RecA−ADP complex did not include magnesium ion or side chains. Here, we have determined the crystal structures of RecA in complex with MgADP and MnAMP−PNP, a nonhydrolyzable analogue of ATP, at resolutions of 1.9 and 2.1 Å, respectively. Both crystals grow in the same conditions and have RecA in a right-handed helical form with a pitch of ∼82 Å. The crystal structures show the detailed interactions of RecA with the nucleotide cofactors, including the metal ion and the γ phosphate of AMP−PNP. There are very few conformational differences between the structures of RecA bound to ADP and AMP−PNP, which differ from uncomplexed RecA only in a slight opening of the P-loop residues 66−73 upon nucleotide binding. To interpret the functional significance of the structure of the MnAMP−PNP complex, a coprotease assay was used to compare the ability of different nucleotides to promote the active, extended conformation of RecA. Whereas ATPγS and ADP−AlF4facilitate a robust coprotease activity, ADP and AMP−PNP do not activate RecA at all. We conclude that the crystal structure of the RecA−MnAMP−PNP complex represents a preisomerization state of the RecA protein that exists after ATP has bound but before the conformational transition to the active state.