Molecular basis of nucleotide-dependent substrate engagement and remodeling by an AAA+ activator.
Molecular basis of nucleotide-dependent substrate engagement and remodeling by an AAA+ activator.
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DOI：
10.1093/nar/gku588
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发表时间：
2014-08
影响因子：
14.9
通讯作者：
Buck M
中科院分区：
文献类型：
--
作者：
Darbari VC;Lawton E;Lu D;Burrows PC;Wiesler S;Joly N;Zhang N;Zhang X;Buck M
AAA+蛋白普遍需要ATP的结合和水解来支持它们的机械力化学作用。在这里，我们通过特异性地改变催化ATP水解所需的Walker B基序，探索ATPase位点在AAA+转录激活蛋白-噬菌体休克蛋白F(PspF)中的作用。其中一个突变体E108Q在三磷酸腺苷的水解过程中存在缺陷，但以三磷酸腺苷依赖的方式完全重塑目标转录复合体，即RNAP-σ54全酶。对E108Q变异体的结构分析表明，与野生型蛋白不同的是，野生型蛋白以ATP和ADP结合的形式与E108残基具有不同的构象，而不同于核苷酸结合，E108Q适应相同的构象。我们的数据表明，E108Q的重塑活性强烈有利于预熔的DNA，利用σ结合与RNAP-ATP54结合足以将失活的全酶转化为活性形式，而本身的水解是导致转录泡形成的核酸重塑所必需的。此外，使用连接的二聚体结构，我们证明了RNAP-DNA54是这种蛋白质重塑活动所必需的，而σ重塑活动可以耐受交替亚基的缺陷水解。
Binding and hydrolysis of ATP is universally required by AAA+ proteins to underpin their mechano-chemical work. Here we explore the roles of the ATPase site in an AAA+ transcriptional activator protein, the phage shock protein F (PspF), by specifically altering the Walker B motif sequence required in catalyzing ATP hydrolysis. One such mutant, the E108Q variant, is defective in ATP hydrolysis but fully remodels target transcription complexes, the RNAP-σ54 holoenzyme, in an ATP dependent manner. Structural analysis of the E108Q variant reveals that unlike wild-type protein, which has distinct conformations for E108 residue in the ATP and ADP bound forms, E108Q adapts the same conformation irrespective of nucleotide bound. Our data show that the remodeling activities of E108Q are strongly favored on pre-melted DNA and engagement with RNAP-σ54 using ATP binding can be sufficient to convert the inactive holoenzyme to an active form, while hydrolysis per se is required for nucleic acid remodeling that leads to transcription bubble formation. Furthermore, using linked dimer constructs, we show that RNAP-σ54 engagement by adjacent subunits within a hexamer are required for this protein remodeling activity while DNA remodeling activity can tolerate defective ATP hydrolysis of alternating subunits.
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影响因子：
16
作者：
Bose, Daniel;Pape, Tillmann;Burrows, Patricia C.;Rappas, Mathieu;Wigneshweraraj, Siva R.;Buck, Martin;Zhang, Xiaodong
通讯作者：
Zhang, Xiaodong
影响因子：
64.5
作者：
Friedman LJ;Gelles J
通讯作者：
Gelles J
影响因子：
5.6
作者：
Burrows, Patricia C.;Wigneshweraraj, Siva R.;Buck, Martin
通讯作者：
Buck, Martin
DOI：
10.1073/pnas.1001188107
发表时间：
2010-05-18
影响因子：
11.1
作者：
Burrows, Patricia C.;Joly, Nicolas;Buck, Martin
通讯作者：
Buck, Martin
影响因子：
16
作者：
Joly, Nicolas;Zhang, Nan;Buck, Martin
通讯作者：
Buck, Martin