Intermediates in SARS-CoV-2 spike-mediated cell entry.

SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.

Intranasal fusion inhibitory lipopeptide prevents direct-contact SARS-CoV-2 transmission in ferrets.

Containment of the COVID-19 pandemic requires reducing viral transmission. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is initiated by membrane fusion between the viral and host cell membranes, which is mediated by the viral spike protein. We have designed lipopeptide fusion inhibitors that block this critical first step of infection and, on the basis of in vitro efficacy and in vivo biodistribution, selected a dimeric form for evaluation in an animal model. Daily intranasal administration to ferrets completely prevented SARS-CoV-2 direct-contact transmission during 24-hour cohousing with infected animals, under stringent conditions that resulted in infection of 100% of untreated animals. These lipopeptides are highly stable and thus may readily translate into safe and effective intranasal prophylaxis to reduce transmission of SARS-CoV-2.

Lipid perturbation by membrane proteins and the lipophobic effect.

Understanding how membrane proteins interact with their environment is fundamental to the understanding of their structure, function and interactions. We have performed coarse-grained molecular dynamics simulations on a series of membrane proteins in a membrane environment to examine the perturbations of the lipids by the presence of protein. We analyze these perturbations in terms of elastic membrane deformations and local lipid protein interactions. However these two factors are insufficient to describe the variety of effects that we observe and the changes caused by membranes proteins to the structure and dynamics of their lipid environment. Other factors that change the conformation available to lipid molecules are evident and are able to modify lipid structure far from the protein surface, and thus mediate long-range interactions between membrane proteins. We suggest that these multiple modifications to lipid behavior are responsible, at the molecular level, for the lipophobic effect we have proposed to account for our observations of membrane protein organization.

Characterization of the motion of membrane proteins using high-speed atomic force microscopy.

For cells to function properly, membrane proteins must be able to diffuse within biological membranes. The functions of these membrane proteins depend on their position and also on protein-protein and protein-lipid interactions. However, so far, it has not been possible to study simultaneously the structure and dynamics of biological membranes. Here, we show that the motion of unlabelled membrane proteins can be characterized using high-speed atomic force microscopy. We find that the molecules of outer membrane protein F (OmpF) are widely distributed in the membrane as a result of diffusion-limited aggregation, and while the overall protein motion scales roughly with the local density of proteins in the membrane, individual protein molecules can also diffuse freely or become trapped by protein-protein interactions. Using these measurements, and the results of molecular dynamics simulations, we determine an interaction potential map and an interaction pathway for a membrane protein, which should provide new insights into the connection between the structures of individual proteins and the structures and dynamics of supramolecular membranes.

Structure of a protein-detergent complex: the balance between detergent cohesion and binding.

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein-detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorin A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.

Single-spanning transmembrane domains in cell growth and cell-cell interactions: More than meets the eye?

As a whole, integral membrane proteins represent about one third of sequenced genomes, and more than 50% of currently available drugs target membrane proteins, often cell surface receptors. Some membrane protein classes, with a defined number of transmembrane (TM) helices, are receiving much attention because of their great functional and pharmacological importance, such as G protein-coupled receptors possessing 7 TM segments. Although they represent roughly half of all membrane proteins, bitopic proteins (with only 1 TM helix) have so far been less well characterized. Though they include many essential families of receptors, such as adhesion molecules and receptor tyrosine kinases, many of which are excellent targets for biopharmaceuticals (peptides, antibodies, et al.). A growing body of evidence suggests a major role for interactions between TM domains of these receptors in signaling, through homo and heteromeric associations, conformational changes, assembly of signaling platforms, etc. Significantly, mutations within single domains are frequent in human disease, such as cancer or developmental disorders. This review attempts to give an overview of current knowledge about these interactions, from structural data to therapeutic perspectives, focusing on bitopic proteins involved in cell signaling.