ATP Is an Allosteric Inhibitor of Coxsackievirus B3 Polymerase.
ATP Is an Allosteric Inhibitor of Coxsackievirus B3 Polymerase.
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DOI：
10.1021/acs.biochem.6b00467
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发表时间：
2016-07-19
期刊：
影响因子：
2.9
通讯作者：
Peersen OB
中科院分区：
文献类型：
--
作者：
Karr JP;Peersen OB
来自正链RNA病毒（如小核糖核酸病毒和黄病毒）的RNA依赖性RNA聚合酶通过在棕榈结构域中独特的NTP诱导的构象变化来关闭其催化活性位点。与完全预先定位的模板核苷酸相结合，这种机制是容易出错的，并导致病毒后代中随机突变的分布，通常被描述为准种。在这里，我们研究了非同源NTPs竞争性抑制柯萨奇病毒B3 3Dpol的单循环延长的程度，3Dpol是一种聚合酶，在病毒感染期间每10 kb合成的RNA产生3-4个突变。使用具有结合2-氨基嘌呤荧光的模板鸟苷的RNA作为延伸报告物，我们发现同源CTP具有24 μM的Km，并且三种非同源核苷酸竞争性地抑制反应，其中GTP的Kic值为500 μM，ATP为1300 μM，UTP为3000 μM。出乎意料的是，ATP也作为一种非竞争性抑制剂，其Kiu为1800 μM，导致3Dpol的变构调节，使聚合酶延伸速率减慢了约4倍。ATP非竞争性抑制需要β-和γ-磷酸盐，并且在两种先前表征的低保真度聚合酶中显著减少。这导致了进一步的突变分析和一个假定的变构结合位点的鉴定，该位点位于保守基序A和D的界面处的NTP进入通道下方，尽管共结晶未能揭示该口袋中结合ATP的任何密度。在病毒的生命周期中的ATP变构效应的潜在作用进行了讨论。
The RNA-dependent RNA polymerases from positive strand RNA viruses, such as picornaviruses and flaviviruses, close their active sites for catalysis via a unique NTP-induced conformational change in the palm domain. Combined with a fully pre-positioned templating nucleotide, this mechanism is error-prone and results in a distribution of random mutations in the viral progeny often described as a quasispecies. Here we examined the extent to which non-cognate NTPs competitively inhibit single cycle elongation by coxsackievirus B3 3Dpol, a polymerase that generates 3–4 mutations per 10 kb of RNA synthesized during viral infection. Using an RNA with a templating guanosine combined with 2-aminopurine fluorescence as a reporter for elongation, we find that the cognate CTP has a Km of 24 μM and the three non-cognate nucleotides competitively inhibit the reaction with Kic values of 500 μM for GTP, 1300 μM for ATP, and 3000 μM for UTP. Unexpectedly, ATP also acted as an uncompetitive inhibitor with a Kiu of 1800 μM, resulting in allosteric modulation of 3Dpol that slowed the polymerase elongation rate ≈4-fold. ATP uncompetitive inhibition required the β- and γ-phosphates and was significantly diminished in two previously characterized low-fidelity polymerases. This led to further mutational analysis and the identification of a putative allosteric binding site below the NTP entry channel at the interface of conserved motifs A and D, although co-crystallization failed to reveal any density for bound ATP in this pocket. The potential role of an ATP allosteric effect during the virus lifecycle is discussed.
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DOI：
10.3390/v7082829
发表时间：
2015-08-06
期刊：
Viruses
影响因子：
--
作者：
Ferrer-Orta C;Ferrero D;Verdaguer N
通讯作者：
Verdaguer N
DOI：
10.1016/j.bbapap.2009.07.021
发表时间：
2010-05
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Joyce CM
通讯作者：
Joyce CM
影响因子：
5.4
作者：
Bressanelli, S;Tomei, L;De Francesco, R
通讯作者：
De Francesco, R
影响因子：
2.9
作者：
Arnold, JJ;Cameron, CE
通讯作者：
Cameron, CE
影响因子：
3.7
作者：
EMINI, EA;SCHLEIF, WA;WIMMER, E
通讯作者：
WIMMER, E