Trapping of a catalytic HIV reverse transcriptase•template:primer complex through a disulfide bond
Trapping of a catalytic HIV reverse transcriptase•template:primer complex through a disulfide bond
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DOI：
10.1016/s1074-5521(00)00113-7
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发表时间：
2000-05-01
影响因子：
--
通讯作者：
Verdine, GL
中科院分区：
文献类型：
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作者：
Huang, HF;Harrison, SC;Verdine, GL
背景资料：HIV-1逆转录酶（RT）是治疗获得性免疫缺陷综合征（AIDS）的主要靶点，然而RT的耐药突变影响了治疗。努力了解RT的酶促机制和抗RT药物的突变抗性的基础已经阻碍了未能结晶催化信息RT-底物complex.Results：我们在这里提出的实验，使我们能够了解未能结晶这样一个复杂的原因。基于这种理解，我们设计了一种新的方法，使用组合二硫键交联策略来捕获催化RT.模板：引物.dNTP三元复合物，从而使共晶体的生长适合于高分辨率的结构分析。晶体包含一个完全组装的活性位点，准备进行催化。交联本身似乎是构象移动的，和周围的区域是不失真的，这表明交联是一个结构上的被动设备，仅仅起到防止解离的催化complex.Conclusions：这里讨论的新策略导致了催化相关的RT的结晶和结构测定。模板：引物。dNTP复合物。这种结构使我们能够在分子水平上分析耐药性的可能原因。这些信息将有助于开发新的核苷类似物抑制剂，这可能有助于规避目前的耐药性。本文所述的共价捕获策略可能适用于结构分析难治的其他蛋白质-DNA复合物。
Background: HIV-1 reverse transcriptase (RT) is a major target for the treatment of acquired immunodeficiency syndrome (AIDS), Resistance mutations in RT compromise treatment, however. Efforts to understand the enzymatic mechanism of RT and the basis for mutational resistance to anti-RT drugs have been hampered by the failure to crystallize a catalytically informative RT-substrate complex.Results: We present here experiments that allow us to understand the reason for the failure to crystallize such a complex. Based on this understanding, we have devised a new approach for using a combinatorial disulfide cross-linking strategy to trap a catalytic RT.template:primer.dNTP ternary complex, thereby enabling the growth of co-crystals suitable for high-resolution structural analysis. The crystals contain a fully assembled active site poised for catalysis. The cross-link itself appears to be conformationally mobile, and the surrounding region is undistorted, suggesting that the cross-link is a structurally passive device that merely acts to prevent dissociation of the catalytic complex.Conclusions: The new strategy discussed here has resulted in the crystallization and structure determination of a catalytically relevant RT.template:primer.dNTP complex. The structure has allowed us to analyze possible causes of drug resistance at the molecular level. This information will assist efforts to develop new classes of nucleoside analog inhibitors, which might help circumvent current resistance profiles. The covalent trapping strategy described here may be useful with other protein-DNA complexes that have been refractory to structural analysis.