Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3027, USA.
J Virol. 2010 Oct;84(19):10311-21. doi: 10.1128/JVI.00688-10. Epub 2010 Jul 21.
The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states, meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrometry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydrogens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange correlated well with alterations in binding surface (P value = 0.0004). For the structurally variable gp120, however, reductions in flexibility extended outside the binding surface, and regions of expected high structural diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse conformations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for understanding how local conformational stability influences entropic change, conformational diversity, and structural rearrangements in the gp120-CD4 binding reaction.
HIV-1 gp120 包膜糖蛋白与 CD4 受体的结合反应涉及焓和熵的异常变化。与此同时，gp120 未配体结合和各种配体结合状态的晶体结构揭示了一个能够折叠成多种构象的内部结构域、一个结构不变的外部结构域，以及在 CD4 结合状态下的一个桥接片小结构域。然而，这些研究仅提供了每个状态灵活性的一些线索。在这里，我们使用酰胺氢/氘交换与质谱联用的方法，提供 HIV-1 gp120 在未配体结合和 CD4 结合状态下局部构象稳定性的定量测量。平均而言，未配体结合的核心 gp120 显示出比具有相同组成的理论上无结构蛋白快 10000 倍的骨架酰胺氢交换率，而 CD4 的结合进一步将 gp120 酰胺交换率降低了 10 倍。对于结构上不变的 CD4，交换的变化与结合表面的变化很好地相关（P 值=0.0004）。然而，对于结构上可变的 gp120，柔韧性的降低扩展到结合表面之外，并且预期具有高结构多样性的区域（内部结构域/桥接片）在未配体状态下的交换速度比低多样性区域快约 20 倍。因此，尽管熵的减少非常显著，但未配体结合的 gp120 或游离的 CD4 都没有明显的无结构，这表明构成 gp120 未配体状态的大部分不同构象都是合理有序的。这些结果为理解局部构象稳定性如何影响熵变、构象多样性以及 gp120-CD4 结合反应中的结构重排提供了一个框架。
