Stoichiometric Analyses of Soluble CD4 to Native-like HIV-1 Envelope by Single-Molecule Fluorescence Spectroscopy
Parul Agrawal,
Anthony L. DeVico,
James S. Foulke, Jr.,
George K. Lewis,
Marzena Pazgier,
Krishanu Ray
Affiliations
Parul Agrawal
Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA
Anthony L. DeVico
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
James S. Foulke, Jr.
Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA
George K. Lewis
Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA; Department of Microbiology and Immunology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA
Marzena Pazgier
Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA; Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA
Krishanu Ray
Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA; Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA; Corresponding author
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
