MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat

Abstract

HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.

Resistance to Second-Generation HIV-1 Maturation Inhibitors

“…While these structures contribute significantly to our understanding of the basis for MI activity and the structural correlates of MI resistance, several key questions remain. In particular, it has been proposed (21,22,52) that the compounds dock inside the six-helix bundle, but at the current resolution, it is not clear how the compounds are oriented within the core of the bundle (with the extended C-28 side chain facing upwards into CA or downwards toward SP1). It is also not apparent what impact MI binding has on the structure of this region.…”

“…Although the two chemical classes of MIs (BVM and its analogs and PF96) act in similar ways, they also exhibit interesting differences in their behaviors: unlike BVM, PF96 activity is not compromised by the SP1-V7A polymorphism, and PF96 but not BVM rescues the assembly defect imposed by the G156E, P157S, and P160L CA MHR mutations (17). As mentioned above, recent structural studies have provided a 3-to 4-Å view of the six-helix bundle that spans the CA-SP1 boundary region (21,22,52). Figure 14 presents a structural model showing a cutaway of the six-helix bundle and the location of mutations selected in subtype B (Fig.…”

“…Recent data obtained by integrated nuclear magnetic resonance (NMR), cryo-electron microscopy (cryo-EM), and molecular dynamics simulations confirm that SP1-T8I stabilizes a helical conformation at the CA-SP1 junction (51). Wagner et al (22) recently speculated that MIs may bind the immature Gag lattice by docking inside a central channel within the six-helix bundle, and density corresponding to BVM was observed at the center of this bundle (21,52).…”

Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking

“…MicroED has been used to solve novel protein and small molecule pharmaceutical crystal structures (de la Cruz et al, 2017; Gruene et al, 2018; Jones et al, 2018; Rodriguez et al, 2015; Xu et al, 2019). Previous ligand-protein interactions determined using MicroED relied on co-crystallization or incubation of the protein and ligand in crystallization drops prior to applying the crystals to the grid (Clabbers et al, 2020; Purdy et al, 2018; Seidler et al, 2018). In this way, novel co-crystals of the HIV-GAG bevirimat complex were determined (Purdy et al, 2018).…”

“…Previous ligand-protein interactions determined using MicroED relied on co-crystallization or incubation of the protein and ligand in crystallization drops prior to applying the crystals to the grid (Clabbers et al, 2020; Purdy et al, 2018; Seidler et al, 2018). In this way, novel co-crystals of the HIV-GAG bevirimat complex were determined (Purdy et al, 2018). The first-generation HIV-I maturation inhibitor bevirimat was found to occupy the six-fold axis of the protein hexamer along the c axis of the unit cell.…”