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

doi:10.1016/j.celrep.2019.08.074

Stoichiometric Analyses of Soluble CD4 to Native-like HIV-1 Envelope by Single-Molecule Fluorescence Spectroscopy

Cell Rep. 2019 Oct 1;29(1):176-186.e4. doi: 10.1016/j.celrep.2019.08.074.

Authors

Parul Agrawal¹, Anthony L DeVico², James S Foulke Jr¹, George K Lewis³, Marzena Pazgier⁴, Krishanu Ray⁵

Affiliations

¹ Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA.
² 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.
³ 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.
⁴ 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.
⁵ 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. Electronic address: kray@som.umaryland.edu.

Abstract

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.

Keywords: HIV-1 Env trimers; association kinetics; cooperativity; ligand binding; single-molecule fluorescence; soluble CD4; stoichiometric analyses.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

CD4 Antigens / immunology*
Cell Line
HIV Infections / immunology
HIV-1 / immunology*
Humans
Ligands
Protein Binding / immunology
Spectrometry, Fluorescence / methods
env Gene Products, Human Immunodeficiency Virus / immunology*

Substances

CD4 Antigens
Ligands
env Gene Products, Human Immunodeficiency Virus

Abstract

Publication types

MeSH terms

Substances

Grants and funding