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.