Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease
Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease
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DOI：
10.1002/pro.2086
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发表时间：
2012-07-01
期刊：
影响因子：
8
通讯作者：
Schiffer, Celia A.
中科院分区：
文献类型：
--
作者：
Alvizo, Oscar;Mittal, Seema;Schiffer, Celia A.
HIV-1 蛋白酶可识别并切割超过 12 种不同的底物，从而导致病毒成熟。虽然这些底物没有保守基序，但它们在病毒生命周期中被蛋白酶专门选择和切割。蛋白酶内发生耐药突变，损害抑制剂结合，但允许持续识别所有这些底物。虽然底物包膜定义了底物识别的一般形状，但成功预测底物结合特异性的决定因素将为抗性蛋白酶中改变分子识别的机制提供更多见解。我们使用正计算设计方法设计了一种特异性改变的 HIV 蛋白酶变体，并使用 X 射线晶体学和酶生物化学验证了该设计。工程变体 Pr3 (A28S/D30F/G48R) 被设计为优先与 HIV 蛋白酶的三分之一天然底物结合； RTRH 优于 p2-NC 和 CA-p2。在动力学测定中，与野生型 (WT) 相比，RTRH 对 Pr3 的结合特异性增加了三倍，这通过等温滴定量热法进一步证实。确定了 WT 蛋白酶的晶体结构以及设计的与 RTRH、CA-p2 和 p2-NC 复合物的变体。对设计的复合物的结构分析表明，其中一种工程取代（G48R）可能稳定异质瓣构象，从而促进底物结合的替代模式。我们的结果表明，虽然可以在 HIV 蛋白酶中设计底物特异性，但蛋白酶的结构柔韧性限制了预测的相互作用的传播。这些结果为 HIV-1 蛋白酶底物结合特异性的可塑性和结构决定因素提供了新的见解。
HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RTRH over p2-NC and CA-p2. In kinetic assays, RTRH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RTRH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.