Structural characterization of the POTRA domains from A. baumannii reveals new conformations in BamA.

Recent studies have demonstrated BamA, the central component of the β-barrel assembly machinery (BAM), as an important therapeutic target to combat infections caused by Acinetobacter baumannii and other Gram-negative pathogens. Homology modeling indicates BamA in A. baumannii consists of five polypeptide transport-associated (POTRA) domains and a β-barrel membrane domain.

Molecular Dynamics Reveals Altered Interactions between Belzutifan and HIF-2 with Natural Variant G323E or Proximal Phosphorylation at T324.

In patients with von-Hippel Lindau (VHL) disease, hypoxia-independent accumulation of HIF-2α leads to increased transcriptional activity of HIF-2α:ARNT that drives cancers such as renal cell carcinoma. Belzutifan, a recently FDA-approved drug, is designed to prevent the transcriptional activity of HIF-2α:ARNT, thereby overcoming the consequences of its unnatural accumulation in VHL-dependent cancers. Emerging evidence suggests that the naturally occurring variant G323E located in the HIF-2α drug binding pocket prevents inhibitory activity of belzutifan analogs, though the mechanism of inhibition remains unclear.

Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter from .

Infections caused by spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane.

Broadening access to small-molecule parameterization with the force field toolkit.

Access to accurate force-field parameters for small molecules is crucial for computational studies of their interactions with proteins. Although a number of general force fields for small molecules exist, e.g.

Mg-dependent mechanism of environmental versatility in a multidrug efflux pump.

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell.

SANS reveals lipid-dependent oligomerization of an intramembrane aspartyl protease from H. volcanii.

Reactions that occur within the lipid membrane involve, at minimum, ternary complexes among the enzyme, substrate, and lipid. For many systems, the impact of the lipid in regulating activity or oligomerization state is poorly understood. Here, we used small-angle neutron scattering (SANS) to structurally characterize an intramembrane aspartyl protease (IAP), a class of membrane-bound enzymes that use membrane-embedded aspartate residues to hydrolyze transmembrane segments of biologically relevant substrates.

Thicket and Mesh: How the Outer Membrane Can Resist Tension Imposed by the Cell Wall.

The cell envelope of Gram-negative bacteria is composed of an outer membrane (OM) and an inner membrane (IM) and a peptidoglycan cell wall (CW) between them. Combined with Braun's lipoprotein (Lpp), which connects the OM and the CW, and numerous membrane proteins that exist in both OM and IM, the cell envelope creates a mechanically stable environment that resists various physical and chemical perturbations to the cell, including turgor pressure caused by the solute concentration difference between the cytoplasm of the cell and the extracellular environment. Previous computational studies have explored how individual components (OM, IM, and CW) can resist turgor pressure although combinations of them have been less well studied.

Enhanced Surface Accessibility of SARS-CoV-2 Omicron Spike Protein Due to an Altered Glycosylation Profile.

SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding in proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics and disulfide bonding of recombinant spike proteins derived from the SARS-CoV-2 Omicron variant (B.1.

Seeing is believing: Illuminating the Gram-negative outer membrane with molecular dynamics simulations.

Recent advances in molecular dynamics (MD) simulations have led to rapid improvement in our understanding of the molecular details of the outer membranes (OMs) of Gram-negative bacteria. In this review, we highlight the latest discoveries from MD simulations of OMs, shedding light on the dynamic nature of these bacteria's first line of defense. With the focus on cutting-edge approaches, we explore the OM's sensitivity to structural features, including divalent cations and membrane composition, which have emerged as crucial determinants of antimicrobial passage.

Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10.

Doa10 (MARCHF6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. Although Doa10 is highly conserved across eukaryotes, it is not understood how Doa10 recognizes its substrates. Here, we define the substrate recognition mechanism of Doa10 by structural and functional analyses on Saccharomyces cerevisiae Doa10 and its model substrates.

Generative β-hairpin design using a residue-based physicochemical property landscape.

De novo peptide design is a new frontier that has broad application potential in the biological and biomedical fields. Most existing models for de novo peptide design are largely based on sequence homology that can be restricted based on evolutionarily derived protein sequences and lack the physicochemical context essential in protein folding. Generative machine learning for de novo peptide design is a promising way to synthesize theoretical data that are based on, but unique from, the observable universe.

From simple to complex: Reconstructing all-atom structures from coarse-grained models using cg2all.

In this issue of Structure, Heo and Feig present cg2all, a novel deep-learning model capable of efficiently predicting all-atom protein structures from coarse-grained (CG) representations. The model maintains high accuracy, even when the CG model is simplified to a single bead per residue, and has a number of promising applications.

Competition between inside-out unfolding and pathogenic aggregation in an amyloid-forming β-propeller.

Studies of folded-to-misfolded transitions using model protein systems reveal a range of unfolding needed for exposure of amyloid-prone regions for subsequent fibrillization. Here, we probe the relationship between unfolding and aggregation for glaucoma-associated myocilin. Mutations within the olfactomedin domain of myocilin (OLF) cause a gain-of-function, namely cytotoxic intracellular aggregation, which hastens disease progression.

A partial human LCK defect causes a T cell immunodeficiency with intestinal inflammation.

Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea.

Drug Binding to BamA Targets Its Lateral Gate.

BamA, the core component of the β-barrel assembly machinery (BAM) complex, is an outer-membrane protein (OMP) in Gram-negative bacteria. Its function is to insert and fold substrate OMPs into the outer membrane (OM). Evidence suggests that BamA follows the asymmetric hybrid-barrel model where the first and last strands of BamA separate, a process known as lateral gate opening, to allow nascent substrate OMP β-strands to sequentially insert and fold through β-augmentation.

Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein.

Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations.

Structural basis of mitochondrial protein import by the TIM23 complex.

Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel.

Understanding Virus Structure and Dynamics through Molecular Simulations.

Viral outbreaks remain a serious threat to human and animal populations and motivate the continued development of antiviral drugs and vaccines, which in turn benefits from a detailed understanding of both viral structure and dynamics. While great strides have been made in characterizing these systems experimentally, molecular simulations have proven to be an essential, complementary approach. In this work, we review the contributions of molecular simulations to the understanding of viral structure, functional dynamics, and processes related to the viral life cycle.

Uncovering the folding mechanism of pertactin: A comparative study of isolated and vectorial folding.

Autotransporters are a large family of virulence factors found in Gram-negative bacteria that play important roles in their pathogenesis. The passenger domain of autotransporters is almost always composed of a large β-helix, with only a small portion of it being relevant to its virulence function. This has led to the hypothesis that the folding of the β-helical structure aids the secretion of the passenger domain across the Gram-negative outer membrane.

Insights into substrate transport and water permeation in the mycobacterial transporter MmpL3.

Mycobacteria, such as Mycobacterium tuberculosis, are characterized by a uniquely thick and waxy cell envelope that consists of two membranes, with a variety of mycolates comprising their outer membrane (OM). The protein Mycobacterial membrane protein Large 3 (MmpL3) is responsible for the transport of a primary OM component, trehalose monomycolate (TMM), from the inner (cytoplasmic) membrane (IM) to the periplasmic space, a process driven by the proton gradient. Although multiple structures of MmpL3 with bound substrates have been solved, the exact pathway(s) for TMM or proton transport remains elusive.