Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA.
Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA; Department of Medicine, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA.
Cell Rep. 2019 Oct 1;29(1):176-186.e4. doi: 10.1016/j.celrep.2019.08.074.
Analyses of HIV-1 envelope (Env) binding to CD4, and the conformational changes the interactions induce, inform the molecular mechanisms and factors governing HIV-1 infection. To address these questions, we used a single-molecule detection (SMD) approach to study the nature of reactions between soluble CD4 (sCD4) and soluble HIV-1 trimers. SMD of these reactions distinguished a mixture of one, two, or three CD4-bound trimer species. Single-ligand trimers were favored at early reaction times and ligand-saturated trimers later. Furthermore, some trimers occupied by one sCD4 molecule did not bind additional ligands, whereas the majority of two ligand-bound species rapidly transitioned to the saturated state. Quantification of liganded trimers observed in reactions with various sCD4 concentrations reflected an overall negative cooperativity in ligand binding. Collectively, our results highlight the general utility of SMD in studying protein interactions and provide critical insights on the nature of sCD4-HIV-1 Env interactions.
对 HIV-1 包膜 (Env) 与 CD4 的结合以及这些相互作用引起的构象变化进行分析，可以深入了解 HIV-1 感染的分子机制和相关因素。为了解决这些问题，我们使用单分子检测 (SMD) 方法研究了可溶性 CD4 (sCD4) 和可溶性 HIV-1 三聚体之间反应的性质。这些反应的 SMD 区分了一种、两种或三种 sCD4 结合三聚体物质的混合物。在早期反应时间，单配体三聚体是优势物种，而配体饱和三聚体则较晚出现。此外，一些被一个 sCD4 分子占据的三聚体不再结合额外的配体，而大多数两个配体结合的物种则迅速过渡到饱和状态。用不同浓度的 sCD4 进行反应时观察到的配体结合三聚体的定量分析反映了配体结合的总体负协同性。总的来说，我们的结果突出了 SMD 在研究蛋白质相互作用中的普遍适用性，并为 sCD4-HIV-1 Env 相互作用的性质提供了重要的见解。
