Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein.

The COVID-19 pandemic has prompted a rapid response in vaccine and drug development. Herein, we modeled a complete membrane-embedded SARS-CoV-2 spike glycoprotein and used molecular dynamics simulations with benzene probes designed to enhance discovery of cryptic pockets. This approach recapitulated lipid and host metabolite binding sites previously characterized by cryo-electron microscopy, revealing likely ligand entry routes, and uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop. A full representation of glycan moieties was essential to accurately describe pocket dynamics. A multi-conformational behavior of the 617-628 loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry experiments, supportive of opening and closing dynamics. The pocket is the site of multiple mutations associated with increased transmissibility found in SARS-CoV-2 variants of concern including Omicron. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.

Manipulating turn residues on de novo designed ß-hairpin peptides for selectivity against drug-resistant bacteria.

Synthetic ß-hairpin antimicrobial peptides (AMPs) offer a useful source for the development of novel antimicrobial agents. ß-hairpin peptides generally consist of two side strands bridged by a reverse turn. In literature, most studies focused on the modifications of the side strands to manipulate the stability and activity of ß-hairpin peptides, and much less is known about the impact of the turn region. By designing a series of de novo ß-hairpin peptides with identical side strands but varied turns, we demonstrated that mutations of only 2 to 4 amino acids at the turn region could impart a wide range of antimicrobial profiles among synthetic ß-hairpin AMPs. BTT2-4 and BTT6 displayed selective potency against Gram-negative bacteria, with minimum inhibitory concentrations (MICs) of 4-8 µM. In contrast, BTT1 exhibited broad-spectrum activity, with MICs of 4-8 µM against both Gram-positive and Gram-negative strains. Additionally, BTT1 was potent against methicillin-resistant Staphylococcus aureus (MRSA) and colistin-resistant Enterobacterales. The antimicrobial potency of BTT1 persisted after 14 days of serial passage. Mechanistic studies revealed that interactions between lipopolysaccharide (LPS) and the peptides were critical to their membranolytic activity against the bacterial inner membrane. Aside from folding stability, we observed that a degree of conformational flexibility was required for disruptive membrane interactions. STATEMENT OF SIGNIFICANCE: By examining the significance of the turn region of ß-hairpin peptides, we present valuable knowledge to the design toolkit of novel antimicrobial peptides as alternative therapeutics to overcome antibiotic resistance. Our de novo designed synthetic peptides displayed selective activity against Gram-negative bacteria and potent activity against clinically relevant antibiotic-resistant strains (e.g. colistin-resistant Enterobacterales and methicillin-resistant Staphylococcus aureus). The bactericidal activity of our peptides was shown to be robust in the presence of proteolytic trypsin and saline, conditions that could suppress peptide activity. Our peptides were also determined to be non-cytotoxic against a human cell line.

The nanotube express: Delivering a stapled peptide to the cell surface.

HYPOTHESIS: Carbon nanotubes (CNTs) represent a novel platform for cellular delivery of therapeutic peptides. Chemically-functionalized CNTs may enhance peptide uptake by improving their membrane targeting properties. EXPERIMENTS: Using coarse-grained (CG) molecular dynamics (MD) simulations, we investigate membrane interactions of a peptide conjugated to pristine and chemically-modified CNTs. As proof of principle, we focus on their interactions with PM2, an amphipathic stapled peptide that inhibits the E3 ubiquitin ligase HDM2 from negatively regulating the p53 tumor suppressor. CNT interaction with both simple planar lipid bilayers as well as spherical lipid vesicles was studied, the latter as a surrogate for curved cellular membranes. FINDINGS: Membrane permeation was rapid and spontaneous for both pristine and oxidized CNTs when unconjugated. This was slowed upon addition of a noncovalently attached peptide surface "sheath", which may be an effective way to slow CNT entry and avert membrane rupture. The CNT conjugates were observed to "desheath" their peptide layer at the bilayer interface upon insertion, leaving their cargo behind in the outer leaflet. This suggests that a synergy may exist to optimize CNT safety whilst enhancing the delivery efficiency of "hitchhiking" therapeutic molecules.

The hitchhiker's guide to the periplasm: Unexpected molecular interactions of polymyxin B1 in E. coli.

The periplasm of Gram-negative bacteria is a complex, highly crowded molecular environment. Little is known about how antibiotics move across the periplasm and the interactions they experience. Here, atomistic molecular dynamics simulations are used to study the antibiotic polymyxin B1 within models of the periplasm, which are crowded to different extents. We show that PMB1 is likely to be able to "hitchhike" within the periplasm by binding to lipoprotein carriers-a previously unreported passive transport route. The simulations reveal that PMB1 forms both transient and long-lived interactions with proteins, osmolytes, lipids of the outer membrane, and the cell wall, and is rarely uncomplexed when in the periplasm. Furthermore, it can interfere in the conformational dynamics of native proteins. These are important considerations for interpreting its mechanism of action and are likely to also hold for other antibiotics that rely on diffusion to cross the periplasm.

Details of hydrophobic entanglement between small molecules and Braun's lipoprotein within the cavity of the bacterial chaperone LolA.

The cell envelope of Gram-negative bacteria is synthesized and maintained via mechanisms that are targets for development of novel antibiotics. Here we focus on the process of moving Braun's lipoprotein (BLP) from the periplasmic space to the outer membrane of E. coli, via the LolA protein. In contrast to current thinking, we show that binding of multiple inhibitor molecules inside the hydrophobic cavity of LolA does not prevent subsequent binding of BLP inside the same cavity. Rather, based on our atomistic simulations we propose the theory that once inhibitors and BLP are bound inside the cavity of LolA, driven by hydrophobic interactions, they become entangled with each other. Our umbrella sampling calculations show that on the basis of energetics, it is more difficult to dislodge BLP from the cavity of LolA when it is uncomplexed compared to complexed with inhibitor. Thus the inhibitor reduces the affinity of BLP for the LolA cavity.

Binding from Both Sides: TolR and Full-Length OmpA Bind and Maintain the Local Structure of the E. coli Cell Wall.

We present a molecular modeling and simulation study of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane, in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed binds peptidoglycan largely through electrostatic interactions. Non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides, respectively, maintain the position of the cell wall even in the absence of Braun's lipoprotein. The charged residues that mediate the cell-wall interactions of TolR in our simulations are conserved across a number of species of gram-negative bacteria.

Braun's Lipoprotein Facilitates OmpA Interaction with the Escherichia coli Cell Wall.

Gram-negative bacteria such as Escherichia coli are protected by a complex cell envelope. The development of novel therapeutics against these bacteria necessitates a molecular level understanding of the structure-dynamics-function relationships of the various components of the cell envelope. We use atomistic MD simulations to reveal the details of covalent and noncovalent protein interactions that link the outer membrane to the aqueous periplasmic region. We show that the Braun's lipoprotein tilts and bends, and thereby lifts the cell wall closer to the outer membrane. Both monomers and dimers of the outer membrane porin OmpA can interact with peptidoglycan in the presence of Braun's lipoprotein, but in the absence of the latter, only dimers of OmpA show a propensity to form contacts with peptidoglycan. Our study provides a glimpse of how the molecular components of the bacterial cell envelope interact with each other to mediate cell wall attachment in E. coli.

Progress in Molecular Dynamics Simulations of Gram-Negative Bacterial Cell Envelopes.

Bacteria are protected by complex molecular architectures known as the cell envelope. The cell envelope is composed of regions with distinct chemical compositions and physical properties, namely, membranes and a cell wall. To develop novel antibiotics to combat pathogenic bacteria, molecular level knowledge of the structure, dynamics, and interplay between the chemical components of the cell envelope that surrounds bacterial cells is imperative. In addition, conserved molecular patterns associated with the bacterial envelope are recognized by receptors as part of the mammalian defensive response to infection, and an improved understanding of bacteria-host interactions would facilitate the search for novel immunotherapeutics. This Perspective introduces an emerging area of computational biology: multiscale molecular dynamics simulations of chemically complex models of bacterial lipids and membranes. We discuss progress to date, and identify areas for future development that will enable the study of aspects of the membrane components that are as yet unexplored by computational methods.