Structure and dynamics of a functional cavity in the HIV-1 Envelope, and its role in conformational changes required for infection Channels and cavities in protein structures often play an important role in modulating structure, function and dynamics. The HIV-1 Envelope (Env) glycoprotein, a conformational machine that utilizes receptor binding mediated conformational changes to effect virus and host cell membrane fusion, is riddled with cavities and channels. A highly conserved cavity ? called the Phe43 cavity - engages the Phe43 residue of receptor CD4 and is critical for CD4-induced Env conformational transitions. The Phe43 cavity is also the binding site of broad and potent antibodies, drugs and peptide inhibitors of HIV-1 entry. The CD4-mimetic miniprotein M48U1, effects broad and potent HIV-1 neutralization, binds HIV-1 gp120 with pM affinity, showed efficacy as a vaginal microbicide in animal models, and more recently, was shown to synergize with the drug Tenofovir to inhibit HIV infection in activated PBMCs and human cervicovaginal histocultures. M48U1 inserts a methoxy cyclohexyl moiety to fill the Phe43 cavity. M48U1 and its analogs are the only class of ligands that reach deep into the Phe43 cavity, a property that makes them valuable tools for probing the structure of a deep, otherwise not easily accessible cavity.We have previously utilized M48U1 and its analogs to probe the structure of the Phe- 43 cavity in gp120 monomers. High resolution structures of M48U1 bound to HIV-1 Env gp120 revealed ligand flexibility, and the ability to fit within and adapt to the Phe43 cavities of diverse HIV-1 isolates with minimal perturbations of the cavity structure are key determinants of activity. In this grant we propose to explore the structure of the Phe43 cavity in the closed HIV-1 Env trimer using M48U1 as a molecular probe. The innovation in this grant derives from our use of M48U1 and its analogs as molecular probes of the Phe43 cavity, from the advances in cryo-EM technology that include improved specimen vitrification methods, improved microscope hardware, automated methods for high-throughput data collection, and advanced algorithms for data processing. These advances have recently allowed us to establish a rapid pipeline for determining high resolution structures of HIV-1 Env complexes. The scientific premise of this grant is that the Phe43 cavity is a critical functional component in HIV-1 Env, central to the activity of the CD4 receptor and many broad and potent antibodies and drugs. M48U1 is an exceptionally effective HIV-1 entry inhibitor, and one of the only ligands that fills the entire Phe-43 cavity. Exploring the structures of HIV-1 Env when bound to M48U1 will provide insights into the structure and the conformational constraints on the cavity in the closed state of the Env.

Public Health Relevance

In this grant we will investigate the structure and dynamics of the conserved HIV-1 Env Phe43 cavity using CD4-mimetic miniprotein M48U1 as a molecular probe. We will determine structures of M48U1 bound to closed and open HIV-1 Env trimers. We will complement structural studies with cell-surface Env binding assays using a fluorescently labeled M48U1, and with neutralization assays. These studies will advance our understanding of the HIV-1 entry mechanism and the function and mechanics of a critical Env cavity.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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HIV Molecular Virology, Cell Biology, and Drug Development Study Section (HVCD)
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Mcdonald, David Joseph
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Duke University
Schools of Medicine
United States
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