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Recent studies have demonstrated BamA, the central component of the ß-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 ß-barrel membrane domain. We characterized the POTRA domains of BamA from A. baumannii in solution using size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) analysis and determined crystal structures in two conformational states that are drastically different than those previously observed in BamA from other bacteria, indicating that the POTRA domains are even more conformationally dynamic than has been observed previously. Molecular dynamics simulations of the POTRA domains from A. baumannii and Escherichia coli allowed us to identify key structural features that contribute to the observed novel states. Together, these studies expand on our current understanding of the conformational plasticity within BamA across differing bacterial species.

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Infections caused by Mycobacterium 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. Here, we focus on MmpL3 from Mycobacterium tuberculosis, an essential inner membrane transporter of the resistance-nodulation-division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro, inside macrophages, and in M. tuberculosis-infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.IMPORTANCEMmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition.

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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. Additionally, studies have provided novel insights into outer-membrane proteins (OMPs), revealing their intricate roles in substrate translocation and their distinct interactions with lipopolysaccharides (LPS) in the OM. Finally, we explore the challenging process of ß-barrel membrane protein insertion, showcasing recent findings that have enhanced our grasp of this fundamental biological phenomenon.

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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.

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Recent studies in polymer physics have created macro-scale analogs to solute microscopic polymer chains like DNA by inducing diffusive motion on a chain of beads. These bead chains have persistence lengths of O(10) links and undergo diffusive motion under random fluctuations like vibration. We present a bead chain model within a new stochastic forcing system: an air fluidizing bed of granular media. A chain of spherical 6 mm resin beads crimped onto silk thread are buffeted randomly by the multiphase flow of grains and low density rising air "bubbles". We "thermalize" bead chains of various lengths at different fluidizing airflow rates, while X-ray imaging captures a projection of the chains' dynamics within the media. With modern 3D printing techniques, we can better represent complex polymers by geometrically varying bead connections and their relative strength, e.g., mimicking the variable stiffness between adjacent nucleotide pairs of DNA. We also develop Discrete Element Method (DEM) simulations to study the 3D motion of the bead chain, where the bead chain is represented by simulated spherical particles connected by linear and angular spring-like bonds. In experiment, we find that the velocity distributions of the beads follow exponential distributions rather than the Gaussian distributions expected from polymers in solution. Through use of the DEM simulation, we find that this difference can likely be attributed to the distributions of the forces imparted onto the chain from the fluidized bed environment. We anticipate expanding this study in the future to explore a wide range of chain composition and confinement geometry, which will provide insights into the physics of large biopolymers.

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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. Here, employing molecular dynamics simulations we investigate putative pathways for either transport species. We hypothesized that MmpL3 will cycle through similar conformational states as the related transporter AcrB, which we used as targets for modeling the conformation of MmpL3. A continuous water pathway through the transmembrane region was found in one of these states, illustrating a putative pathway for protons. Additional equilibrium simulations revealed that TMM can diffuse from the membrane into a binding pocket in MmpL3 spontaneously. We also found that acetylation of TMM, which is required for transport, makes it more stable within MmpL3's periplasmic cavity compared with the unacetylated form.

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Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is one of the leading causes of death in developing countries. Non-tuberculous mycobacteria (NTM) infections are rising and prey upon patients with structural lung diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. All mycobacterial infections require lengthy treatment regimens with undesirable side effects. Therefore, new antimycobacterial compounds with novel mechanisms of action are urgently needed. Published indole-2-carboxamides (IC) with suggested inhibition of the essential transporter MmpL3 showed good potency against whole-cell M.tb, yet had poor aqueous solubility. This project focused on retaining the required MmpL3 inhibitory pharmacophore and increasing the molecular heteroatom percentage by reducing lipophilic atoms. We evaluated pyrrole, mandelic acid, imidazole, and acetamide functional groups coupled to lipophilic head groups, where lead acetamide-based compounds maintained high potency against mycobacterial pathogens, had improved in vitro ADME profiles over their indole-2-carboxamide analogs, were non-cytotoxic, and were determined to be MmpL3 inhibitors.

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