Specific protein-lipid interactions are critical for viral assembly. We present a molecular dynamics simulation study on the binding mechanism of the membrane targeting domain of HIV-1 Gag protein. The matrix (MA) domain drives Gag onto the plasma membrane through electrostatic interactions at its highly-basic-region (HBR), located near the myristoylated (Myr) N-terminus of the protein. Our study suggests Myr insertion is involved in the sorting of membrane lipids around the protein binding site to prepare it for viral assembly. Our realistic membrane models confirm interactions with PIP2 and PS lipids are highly favored around the HBR, and are strong enough to keep the protein bound even without Myr insertion. We characterized Myr insertion events from microsecond trajectories, and examined the membrane response upon initial membrane targeting by MA. Insertion events only occur with one of the membrane models, showing a combination of surface charge and internal membrane structure modulate this process.
The simulation trajectories used for the analysis presented in this work are available at the Pittsburgh Supercomputing Center (PSC) Database for simulations run on the Anton2 Machine (http://psc.edu/anton-project-summaries?id=3071&pid=34).
All-atom molecular dynamics simulations of the matrix domain of HIV-1 Gag protein and model membranesPittsburgh Supercomputing Center Public Repository.
- Viviana Monje-Galvan
- Gregory A Voth
- Gregory A Voth
- Gregory A Voth
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- José D Faraldo-Gómez, National Heart, Lung and Blood Institute, National Institutes of Health, United States
© 2020, Monje-Galvan & Voth
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin’s voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl− anion at a conserved binding site formed by the amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen–deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl− binding. This event shortens the TM3–TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin’s mechanical expansion. We observe helix fraying at prestin’s anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin’s fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and help define prestin’s unique voltage-sensing mechanism and electromotility.
The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.