The voltage sensor (VS) domain in Hv1 proton channels mediates a voltage-dependent and H⁺-selective “aqueous” conductance (G_AQ) that is potently modulated by extracellular Zn²⁺. Although two conserved His residues are required for Zn²⁺ effects on G_AQ gating, the atomic structure of the Zn²⁺ coordination site and mechanism by which extracellular Zn²⁺ stabilizes a closed-state conformation remain unknown. Here we use His mutagenesis to identify residues that increase Zn²⁺ potency and are therefore likely to participate in first solvation shell interactions with Zn²⁺. Experimental Zn²⁺-mapping data were then used to constrain the structure of a new resting-state Hv1 model (Hv1 F). Molecular dynamics (MD) simulations show how protein and water atoms directly contribute to octahedral Zn²⁺ coordination spheres in Zn²⁺-bound and -unbound Hv1 F models. During MD simulations, we observed correlated movements of Zn²⁺-interacting side chains and residues in a highly conserved intracellular Coulombic network (ICN) that contains highly conserved Arg “gating charges” in S4 as well as acidic “counter-charges” in S2 and S3 and is known to control VS activation, suggesting that occupancy of the extracellular Zn²⁺ site is conformationally coupled to reorganization of the ICN. To test this hypothesis, we neutralized an ICN Glu residue (E153) and show that in addition to shifting G_AQ activation to more negative voltages, E153A also decreases Zn²⁺ potency. We speculate that extracellular gating-modifier toxins and other ligands may use a generally similar long-range conformational coupling mechanism to modulate VS activation in related ion channel proteins.

The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although remarkable progress has been made in understanding the structures of various Env conformations, microsecond timescale dynamics have not been studied experimentally. Here, we used time-resolved, temperature-jump small-angle x-ray scattering to monitor structural rearrangements in an HIV-1 Env SOSIP ectodomain construct with microsecond precision. In two distinct Env variants, we detected a transition that correlated with known Env structure rearrangements with a time constant in the hundreds of microseconds range. A previously unknown structural transition was also observed, which occurred with a time constant below 10 μs, and involved an order-to-disorder transition in the trimer apex. Using this information, we engineered an Env SOSIP construct that locks the trimer in the prefusion closed state by connecting adjacent protomers via disulfides. Our findings show that the microsecond timescale structural dynamics play an essential role in controlling the Env conformation with impacts on vaccine design.

The HIV-1 envelope glycoprotein (Env) mediates host cell fusion and is the primary target for HIV-1 vaccine design. The Env undergoes a series of functionally important conformational rearrangements upon engagement of its host cell receptor, CD4. As the sole target for broadly neutralizing antibodies, our understanding of these transitions plays a critical role in vaccine immunogen design. Here, we review available experimental data interrogating the HIV-1 Env conformation and detail computational efforts aimed at delineating the series of conformational changes connecting these rearrangements. These studies have provided a structural mapping of prefusion closed, open, and transition intermediate structures, the allosteric elements controlling rearrangements, and state-to-state transition dynamics. The combination of these investigations and innovations in molecular modeling set the stage for advanced studies examining rearrangements at greater spatial and temporal resolution.