Modeling membranes in situ.

Molecular dynamics simulations of cellular membranes have come a long way-from simple model lipid bilayers to multicomponent systems capturing the crowded and complex nature of real cell membranes. In this opinionated minireview, we discuss the current challenge to simulate the dynamics of membranes in their native environment, in situ, with the prospect of reaching the level of whole cells and cell organelles using an integrative modeling framework.

Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex.

Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.

PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection.

Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial1-4. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR-Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. Its robust activity extended to other highly pathogenic coronaviruses, was functionally conserved in bats and mice, and interfered with the uptake of SARS-CoV-2 in both the endocytic and the TMPRSS2-dependent fusion routes. Whole-cell 4Pi single-molecule switching nanoscopy together with bipartite nano-reporter assays found that PLSCR1 directly targeted SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape. A PLSCR1 C-terminal ß-barrel domain-but not lipid scramblase activity-was essential for this fusogenic blockade. Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people3,4, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol.

Supramolecular organization and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

Interrogation of the Pathogen Box reveals small molecule ligands against the mycobacterial trehalose transporter LpqY-SugABC.

Tuberculosis, caused by Mycobacterium tuberculosis, claims ∼1.5 million lives annually. Effective chemotherapy is essential to control TB, however the emergence of drug-resistant strains of TB have seriously threatened global attempts to control and eradicate this deadly pathogen. Trehalose recycling via the LpqY-SugABC importer is essential for the virulence and survival of Mtb and inhibiting or hijacking this transport system is an attractive approach for the development of novel anti-tubercular and diagnostic agents. Therefore, we interrogated the drug-like compounds in the open-source Medicines for Malaria Pathogen Box and successfully identified seven compounds from the TB, kinetoplastids and reference compound disease sets that recognise LpqY. The molecules have diverse chemical scaffolds, are not specific trehalose analogues, and may be used as novel templates to facilitate the development of therapeutics that kill Mtb with a novel mechanism of action via the mycobacterial trehalose LpqY-SugABC transport system.

Peptidoglycan biosynthesis is driven by lipid transfer along enzyme-substrate affinity gradients.

Maintenance of bacterial cell shape and resistance to osmotic stress by the peptidoglycan (PG) renders PG biosynthetic enzymes and precursors attractive targets for combating bacterial infections. Here, by applying native mass spectrometry, we elucidate the effects of lipid substrates on the PG membrane enzymes MraY, MurG, and MurJ. We show that dimerization of MraY is coupled with binding of the carrier lipid substrate undecaprenyl phosphate (C55-P). Further, we demonstrate the use of native MS for biosynthetic reaction monitoring and find that the passage of substrates and products is controlled by the relative binding affinities of the different membrane enzymes. Overall, we provide a molecular view of how PG membrane enzymes convey lipid precursors through favourable binding events and highlight possible opportunities for intervention.

Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter.

The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodeling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen, it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, saturation transfer difference NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the synthesis of trehalose analogs. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogs and specifies a framework that can be exploited for the design of new antitubercular agents and/or diagnostic tools.