| Summary: | Summary: 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. : Agrawal et al. use single-molecule fluorescence approaches to evaluate the interactions between sCD4 and soluble near-native trimer. Their findings highlight the general utility of single-molecule detection in studying protein interactions and provide critical insights into sCD4-Env interaction for better understanding the complex biology of HIV-1 entry into cells. Keywords: single-molecule fluorescence, HIV-1 Env trimers, soluble CD4, stoichiometric analyses, cooperativity, ligand binding, association kinetics
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