Structural basis for the D-stereoselectivity of human DNA polymerase β.
Structural basis for the D-stereoselectivity of human DNA polymerase β.
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DOI：
10.1093/nar/gkx252
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发表时间：
2017-06-02
影响因子：
14.9
通讯作者：
Suo Z
中科院分区：
文献类型：
--
作者：
Vyas R;Reed AJ;Raper AT;Zahurancik WJ;Wallenmeyer PC;Suo Z
具有L-立体化学的核苷逆转录酶抑制剂（NRTI）长期以来一直是病毒感染的有效治疗，因为相对于病毒逆转录酶，人DNA聚合酶表现出强的D-立体选择性。DNA聚合酶的D-立体选择性只是最近才在结构上进行了探索，迄今为止研究的所有三种DNA聚合酶都表现出独特的立体化学选择机制。在这里，我们已经解决了人DNA聚合酶β（hPolβ）的结构，与单核苷酸缺口DNA和L-核苷酸的复合物，并进行预稳态动力学分析，以确定hPolβ的D-立体选择性机制。除了在其他研究中观察到的类似的L-核苷酸核糖环的180°旋转之外，hPolβ、DNA和L-dCTP或抗病毒药物拉米夫定（（-）3 TC-TP）和恩曲他滨（（-）FTC-TP）的三磷酸形式的催化前三元晶体结构几乎没有提供结构证据表明hPolβ遵循先前表征的D-立体选择性机制。相反，hPolβ通过几个活性位点重排的积累来区分L-立体化学，这些活性位点重排导致核苷酸结合亲和力和掺入率降低。这两种NRTI通过碱基和糖修饰逃避了一些活性位点选择，但通过hPolβ无法完成拇指结构域闭合而被选择。
Nucleoside reverse transcriptase inhibitors (NRTIs) with L-stereochemistry have long been an effective treatment for viral infections because of the strong D-stereoselectivity exhibited by human DNA polymerases relative to viral reverse transcriptases. The D-stereoselectivity of DNA polymerases has only recently been explored structurally and all three DNA polymerases studied to date have demonstrated unique stereochemical selection mechanisms. Here, we have solved structures of human DNA polymerase β (hPolβ), in complex with single-nucleotide gapped DNA and L-nucleotides and performed pre-steady-state kinetic analysis to determine the D-stereoselectivity mechanism of hPolβ. Beyond a similar 180° rotation of the L-nucleotide ribose ring seen in other studies, the pre-catalytic ternary crystal structures of hPolβ, DNA and L-dCTP or the triphosphate forms of antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) provide little structural evidence to suggest that hPolβ follows the previously characterized mechanisms of D-stereoselectivity. Instead, hPolβ discriminates against L-stereochemistry through accumulation of several active site rearrangements that lead to a decreased nucleotide binding affinity and incorporation rate. The two NRTIs escape some of the active site selection through the base and sugar modifications but are selected against through the inability of hPolβ to complete thumb domain closure.