Recombinant HIV type 1 gp41 (Bal) (a.a. 537-676) [His]

Cryo-electron microscopy and X-ray crystallography have shown that the pre- and postfusion states of the HIV-1 gp41 viral coat protein, although very different from one another, each adopt C-3 symmetric structures. A stable homotrimeric structure for the transmembrane domain (TM) also was modeled and supported by experimental data. For a C-3 symmetric structure, alignment in an anisotropic medium must be axially symmetric, with the unique axis of the alignment tensor coinciding with the C-3 axis. However, NMR residual dipolar couplings (RDCs) measured under three different alignment conditions were found to be incompatible with C-3 symmetry. Subsequent measurements by paramagnetic relaxation enhancement, analytical ultracentrifugation, and DEER EPR, indicate that the transmembrane domain is monomeric. N-15 NMR relaxation data and RDCs show that TM is highly ordered and uninterrupted for a total length of 32 residues, extending well into the membrane proximal external region.

The Dual-Action Virolytic Entry Inhibitors, or DAVEI's, are a class of recombinant fusions of a lectin, a linker polypeptide, and a 15-residue fragment from the membrane-proximal external region (MPER) of HIV-1 gp41. DAVEI's trigger rupture of HIV-1 virions, and the interaction site between DAVEI MPER and HIV-1 lies in the gp41 component of the envelope glycoprotein Env. Here, we explore the hypothesis that DAVEI MPER engages Env gp41 in a mode structurally similar to a crystallographic MPER trimer. We used alchemical free-energy perturbation to assess the thermodynamic roles of each of the four conserved tryptophan residues on each protomer of MPER3. We found that a W666A mutation had a large positive G for all three protomers, while W672A had a large positive G for only two of the three protomers, with the other tryptophans remaining unimportant contributors to MPER3 stability. The protomer for which W672 is not important is unique in the placement of its W666 sidechain between the other two protomers. We show that the unique orientation of this W666 sidechain azimuthally rotates its protomer away from the orientation it would have if the trimer were symmetric, resulting in the diminished interaction of this W672 with the rest of MPER3. Our findings are consistent with our previous experimental study of W-to-A mutants of DAVEI. This suggests that DAVEI MPER may engage HIV-1 Env to form a mixed trimer state in which one DAVEI MPER forms a trimer by displacing a more weakly interacting protomer of the endogenous Env MPER trimer.