Identification of the SSB binding site on E. coli RecQ reveals a conserved surface for binding SSB's C terminus.
Identification of the SSB binding site on E. coli RecQ reveals a conserved surface for binding SSB's C terminus.
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DOI：
10.1016/j.jmb.2008.12.065
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发表时间：
2009-02-27
影响因子：
5.6
通讯作者：
Keck JL
中科院分区：
文献类型：
--
作者：
Shereda RD;Reiter NJ;Butcher SE;Keck JL
RecQ DNA解旋酶与异源伴侣蛋白一起催化DNA代谢活动，包括重组起始和停止复制叉加工。对于典型的大肠杆菌RecQ蛋白，与单链DNA结合（SSB）蛋白的直接相互作用刺激其DNA解绕活性。RecQ和SSB之间的复合物形成是由RecQ翼螺旋结构域（WH）介导的，该结构域结合了SSB的9个c端大多数残基，这是一个高度保守的序列，称为SSB- ct元件。通过核磁共振和突变分析，我们确定了大肠杆菌RecQ上的SSB-Ct结合袋。该结合位点与先前鉴定的大肠杆菌外切酶I上的SSB-Ct结合位点具有惊人的静电相似性，尽管这两种蛋白中的ssb结合域在结构上没有其他相关。改变与SSB- ct结合有关的RecQ残基的取代会损害RecQ与SSB和SSB/DNA核蛋白复合物的结合。这些替换也降低了变体中SSB刺激的DNA解旋酶活性，尽管RecQ变体中额外的生化变化表明WH结构域在SSB结合之外的解旋酶活性中起作用。在没有DNA的情况下，SSB- ct元件的序列变化足以消除与RecQ的相互作用，并降低RecQ在SSB/DNA底物上的结合和解旋酶活性。这些结果支持了一个模型，在这个模型中，RecQ进化出了一个SSB- ct结合位点，作为一种适应，有助于其在SSB/DNA核蛋白底物上的细胞功能。
RecQ DNA helicases act in conjunction with heterologous partner proteins to catalyze DNA metabolic activities that include recombination initiation and stalled replication fork processing. For the prototypical Escherichia coli RecQ protein, direct interaction with single-stranded DNA-binding (SSB) protein stimulates its DNA unwinding activity. Complex formation between RecQ and SSB is mediated by the RecQ winged-helix domain (WH), which binds the nine C-terminal-most residues of SSB, a highly conserved sequence known as the SSB-Ct element. Using nuclear magnetic resonance and mutational analyses, we identify the SSB-Ct binding pocket on E. coli RecQ. The binding site shares striking electrostatic similarity with the previously identified SSB-Ct binding site on E. coli Exonuclease I, although the SSB-binding domains in the two proteins are not otherwise related structurally. Substitutions that alter RecQ residues implicated in SSB-Ct binding impair RecQ binding to SSB and SSB/DNA nucleoprotein complexes. These substitutions also diminish SSB-stimulated DNA helicase activity in the variants, although additional biochemical changes in the RecQ variants indicate a role for the WH domain in helicase activity beyond SSB binding. Sequence changes in the SSB-Ct element are sufficient to abolish interaction with RecQ in the absence of DNA and to diminish RecQ binding and helicase activity on SSB/DNA substrates. These results support a model in which RecQ has evolved an SSB-Ct binding site on its winged-helix domain as an adaptation that aids its cellular functions on SSB/DNA nucleoprotein substrates.
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