The Name Is Barrel, ß-Barrel.

ß-barrels are a class of membrane proteins made up of a cylindrical, anti-parallel ß-sheet with a hydrophobic exterior and a hydrophilic interior. The majority of proteins found in the outer membranes (OMs) of Gram-negative bacteria, mitochondria, and chloroplasts are ß-barrel outer membrane proteins (OMPs). ß-barrel OMPs have a diverse repertoire of functions, including nutrient transport, secretion, bacterial virulence, and enzymatic activity. Here, we discuss the broad functional classes of ß-barrel OMPs, how they are folded into the membrane, and the future of ß-barrel OMP research and its applications.

Energization of Outer Membrane Transport by the ExbB ExbD Molecular Motor.

The outer membranes (OM) of Gram-negative bacteria contain a class of proteins (TBDTs) that require energy for the import of nutrients and to serve as receptors for phages and protein toxins. Energy is derived from the proton motif force (pmf) of the cytoplasmic membrane (CM) through the action of three proteins, namely, TonB, ExbB, and ExbD, which are located in the CM and extend into the periplasm. The leaky phenotype of exbB exbD mutants is caused by partial complementation by homologous tolQ tolR. TonB, ExbB, and ExbD are genuine components of an energy transmission system from the CM into the OM. Mutant analyses, cross-linking experiments, and most recently X-ray and cryo-EM determinations were undertaken to arrive at a model that describes the energy transfer from the CM into the OM. These results are discussed in this paper. ExbB forms a pentamer with a pore inside, in which an ExbD dimer resides. This complex harvests the energy of the pmf and transmits it to TonB. TonB interacts with the TBDT at the TonB box, which triggers a conformational change in the TBDT that releases bound nutrients and opens the pore, through which nutrients pass into the periplasm. The structurally altered TBDT also changes the interactions of its periplasmic signaling domain with anti-sigma factors, with the consequence being that the sigma factors initiate transcription.

Mechanistic insights into fungal mitochondrial outer membrane protein biogenesis.

The majority of mitochondrial proteins are nuclear-encoded and need to be transported into the mitochondria, including the proteins in the outer mitochondrial membrane. For ß-barrel proteins, the preproteins are initially recognized and imported by the TOM complex, then shuttled to the SAM complex via small Tim proteins. For ⍺-helical proteins, some preproteins are recognized by the TOM complex and imported into the membrane by the MIM complex. In recent years multiple structures of the TOM complex and the SAM complex have been reported, increasing our understanding of the mechanism of protein biogenesis in the outer mitochondrial membrane.

The Ton Motor.

The Ton complex is a molecular motor at the inner membrane of Gram-negative bacteria that uses a proton gradient to apply forces on outer membrane (OM) proteins to permit active transport of nutrients into the periplasmic space. Recently, the structure of the ExbB-ExbD subcomplex was determined in several bacterial species, but the complete structure and stoichiometry of TonB have yet to be determined. The C-terminal end of TonB is known to cross the periplasm and interact with TonB-dependent outer membrane transport proteins with high affinity. Yet despite having significant knowledge of these transport proteins, it is not clear how the Ton motor opens a pathway across the outer membrane for nutrient import. Additionally, the mechanism by which energy is harnessed from the inner membrane subcomplex and transduced to the outer membrane via TonB is not well understood. In this review, we will discuss the gaps in the knowledge about the complete structure of the Ton motor complex and the relationship between ion flow used to generate mechanical work at the outer membrane and the nutrient transport process.

The Role of Voltage-Dependent Anion Channel in Mitochondrial Dysfunction and Human Disease.

The voltage-dependent anion channel (VDAC) is a ß-barrel membrane protein located in the outer mitochondrial membrane (OMM). VDAC has two conductance states: an open anion selective state, and a closed and slightly cation-selective state. VDAC conductance states play major roles in regulating permeability of ATP/ADP, regulation of calcium homeostasis, calcium flux within ER-mitochondria contact sites, and apoptotic signaling events. Three reported structures of VDAC provide information on the VDAC open state via X-ray crystallography and nuclear magnetic resonance (NMR). Together, these structures provide insight on how VDAC aids metabolite transport. The interaction partners of VDAC, together with the permeability of the pore, affect the molecular pathology of diseases including Parkinson's disease (PD), Friedreich's ataxia (FA), lupus, and cancer. To fully address the molecular role of VDAC in disease pathology, major questions must be answered on the structural conformers of VDAC. For example, further information is needed on the structure of the closed state, how binding partners or membrane potential could lead to the open/closed states, the function and mobility of the N-terminal α-helical domain of VDAC, and the physiological role of VDAC oligomers. This review covers our current understanding of the various states of VDAC, VDAC interaction partners, and the roles they play in mitochondrial regulation pertaining to human diseases.

A Biochemical and Structural Understanding of TOM Complex Interactions and Implications for Human Health and Disease.

The central role mitochondria play in cellular homeostasis has made its study critical to our understanding of various aspects of human health and disease. Mitochondria rely on the translocase of the outer membrane (TOM) complex for the bulk of mitochondrial protein import. In addition to its role as the major entry point for mitochondrial proteins, the TOM complex serves as an entry pathway for viral proteins. TOM complex subunits also participate in a host of interactions that have been studied extensively for their function in neurodegenerative diseases, cardiovascular diseases, innate immunity, cancer, metabolism, mitophagy and autophagy. Recent advances in our structural understanding of the TOM complex and the protein import machinery of the outer mitochondrial membrane have made structure-based therapeutics targeting outer mitochondrial membrane proteins during mitochondrial dysfunction an exciting prospect. Here, we describe advances in understanding the TOM complex, the interactome of the TOM complex subunits, the implications for the development of therapeutics, and our understanding of the structure/function relationship between components of the TOM complex and mitochondrial homeostasis.

Protein import and export across the bacterial outer membrane.

The bacterial outer membrane forms an impermeable barrier to the environment, but a wide variety of substances must cross it without compromising the membrane. Perhaps, the most fascinating transport phenomenon is the import and export of very large protein toxins using relatively small ß-barrel proteins residing in the outer membrane. Progress has been made on three systems in recent years that shed light on this process. In this review, we summarize bacteriocin (toxin) import using TonB-dependent transporters and protein secretion by autotransporters and two partner secretion systems.

Building Better Barrels - ß-barrel Biogenesis and Insertion in Bacteria and Mitochondria.

ß-barrel proteins are folded and inserted into outer membranes by multi-subunit protein complexes that are conserved across different types of outer membranes. In Gram-negative bacteria this complex is the barrel-assembly machinery (BAM), in mitochondria it is the sorting and assembly machinery (SAM) complex, and in chloroplasts it is the outer envelope protein Oep80. Mitochondrial ß-barrel precursor proteins are translocated from the cytoplasm to the intermembrane space by the translocase of the outer membrane (TOM) complex, and stabilized by molecular chaperones before interaction with the assembly machinery. Outer membrane bacterial BamA interacts with four periplasmic accessory proteins, whereas mitochondrial Sam50 interacts with two cytoplasmic accessory proteins. Despite these major architectural differences between BAM and SAM complexes, their core proteins, BamA and Sam50, seem to function the same way. Based on the new SAM complex structures, we propose that the mitochondrial ß-barrel folding mechanism follows the budding model with barrel-switching aiding in the release of new barrels. We also built a new molecular model for Tom22 interacting with Sam37 to identify regions that could mediate TOM-SAM supercomplex formation.

Ton motor complexes.

The Ton complex is a molecular motor that uses the proton gradient at the inner membrane of Gram-negative bacteria to apply forces on outer membrane proteins, allowing active transport of nutrients into the periplasmic space. For decades, contradictory data has been reported on the structure and stoichiometry of the Ton complex. However, recent reports strongly support a subunit stoichiometry of 5:2 for the ExbB-ExbD subcomplex. In this review, we summarize the recent discoveries of the structures and proposed mechanisms of the Ton system, as well as similar protein motor complexes in Gram-negative bacteria.

In-depth interrogation of protein thermal unfolding data with MoltenProt.

Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature Tm , should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X-ray crystallography and cryo-electron microscopy, screening for small-molecule binding partners and comparison of effects of point mutations.

Author Correction: Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural insight into toxin secretion by contact-dependent growth inhibition transporters.

Bacterial contact-dependent growth inhibition (CDI) systems use a type Vb secretion mechanism to export large CdiA toxins across the outer membrane by dedicated outer membrane transporters called CdiB. Here, we report the first crystal structures of two CdiB transporters from Acinetobacter baumannii and Escherichia coli. CdiB transporters adopt a TpsB fold, containing a 16-stranded transmembrane ß-barrel connected to two periplasmic domains. The lumen of the CdiB pore is occluded by an N-terminal α-helix and the conserved extracellular loop 6; these two elements adopt different conformations in the structures. We identified a conserved DxxG motif located on strand ß1 that connects loop 6 through different networks of interactions. Structural modifications of DxxG induce rearrangement of extracellular loops and alter interactions with the N-terminal α-helix, preparing the system for α-helix ejection. Using structural biology, functional assays, and molecular dynamics simulations, we show how the barrel pore is primed for CdiA toxin secretion.

Structural insight into mitochondrial ß-barrel outer membrane protein biogenesis.

In mitochondria, ß-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold ß-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane ß-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 ß-barrel opens a lateral gate to accommodate its substrates.

Cloning and Multi-Subunit Expression of Mitochondrial Membrane Protein Complexes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a useful eukaryotic expression system for mitochondrial membrane proteins due to its ease of growth and ability to provide a native membrane environment. The development of the pBEVY vector system has further increased the potential of S. cerevisiae as an expression system by creating a method for expressing multiple proteins simultaneously. This vector system is amenable to the expression and purification of multi-subunit protein complexes. Here we describe the cloning, yeast transformation, and co-expression of multi-subunit outer mitochondrial membrane complexes using the pBEVY vector system.

Structure and Stoichiometry of the Ton Molecular Motor.

The Ton complex is a molecular motor that uses the proton gradient at the inner membrane of Gram-negative bacteria to generate force and movement, which are transmitted to transporters at the outer membrane, allowing the entry of nutrients into the periplasmic space. Despite decades of investigation and the recent flurry of structures being reported by X-ray crystallography and cryoEM, the mode of action of the Ton molecular motor has remained elusive, and the precise stoichiometry of its subunits is still a matter of debate. This review summarizes the latest findings on the Ton system by presenting the recently reported structures and related reports on the stoichiometry of the fully assembled complex.

Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry.

The TonB-ExbB-ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB-ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function.

Hitting with a BAM: Selective Killing by Lectin-Like Bacteriocins.

Lectin-like bacteriocins (LlpAs) are secreted by proteobacteria and selectively kill strains of their own or related species, and they are composed of two B-lectin domains with divergent sequences. In Pseudomonas spp., initial binding of these antibacterial proteins to cells is mediated by the carboxy-terminal domain through d-rhamnose residues present in the common polysaccharide antigen of their lipopolysaccharide, whereas the amino-terminal domain accounts for strain selectivity of killing. Here, we show that spontaneous LlpA-resistant mutants carry mutations in one of three surface-exposed moieties of the essential ß-barrel outer membrane protein insertase BamA, the core component of the BAM complex. Polymorphism of this loop in different Pseudomonas groups is linked to LlpA susceptibility, and targeted cells all share the same signature motif in this loop. Since heterologous expression of such a bamA gene confers LlpA susceptibility upon a resistant strain, BamA represents the primary bacteriocin selectivity determinant in pseudomonads. Contrary to modular bacteriocins that require uptake via the Tol or Ton system, parasitism of BamA as an LlpA receptor advocates a novel bacteriocin killing mechanism initiated by impairment of the BAM machinery.IMPORTANCE Bacteria secrete a variety of molecules to eliminate microbial rivals. Bacteriocins are a pivotal group of peptides and proteins that assist in this fight, specifically killing related bacteria. In Gram-negative bacteria, these antibacterial proteins often comprise distinct domains for initial binding to a target cell's surface and subsequent killing via enzymatic or pore-forming activity. Here, we show that lectin-like bacteriocins, a family of bacteriocins that lack the prototypical modular toxin architecture, also stand out by parasitizing BamA, the core component of the outer membrane protein assembly machinery. A particular surface-exposed loop of BamA, critical for its function, serves as a key discriminant for cellular recognition, and polymorphisms in this loop determine whether a strain is susceptible or immune to a particular bacteriocin. These findings suggest a novel mechanism of contact-dependent killing that does not require cellular uptake. The evolutionary advantage of piracy of an essential cellular compound is highlighted by the observation that contact-dependent growth inhibition, a distinct antagonistic system, can equally take advantage of this receptor.

The ColM Family, Polymorphic Toxins Breaching the Bacterial Cell Wall.

Bacteria host an arsenal of antagonism-mediating molecules to combat for ecologic space. Bacteriocins represent a pivotal group of secreted antibacterial peptides and proteins assisting in this fight, mainly eliminating relatives. Colicin M, a model for peptidoglycan-interfering bacteriocins in Gram-negative bacteria, appears to be part of a set of polymorphic toxins equipped with such a catalytic domain (ColM) targeting lipid II. Diversifying recombination has enabled parasitism of different receptors and has also given rise to hybrid bacteriocins in which ColM is associated with another toxin module. Remarkably, ColM toxins have recruited a diverse array of immunity partners, comprising cytoplasmic membrane-associated proteins with different topologies. Together, these findings suggest that different immunity mechanisms have evolved for ColM, in contrast to bacteriocins with nuclease activities.

Structure of voltage-dependent anion channel-tethered bilayer lipid membranes determined using neutron reflectivity.

Neutron reflectivity (NR) has emerged as a powerful technique to study the structure and behavior of membrane proteins at planar lipid interfaces. Integral membrane proteins (IMPs) remain a significant challenge for NR owing to the difficulty of forming complete bilayers with sufficient protein density for scattering techniques. One strategy to achieve high protein density on a solid substrate is the capture of detergent-stabilized, affinity-tagged IMPs on a nitrilotriacetic acid (NTA)-functionalized self-assembled monolayer (SAM), followed by reconstitution into the lipids of interest. Such protein-tethered bilayer lipid membranes (ptBLMs) have the notable advantage of a uniform IMP orientation on the substrate. Here, NR is used to provide a structural characterization of the ptBLM process from formation of the SAM to capture of the detergent-stabilized IMP and lipid reconstitution. The mitochondrial outer-membrane voltage-dependent anion channel (VDAC), which controls the exchange of bioenergetic metabolites between mitochondria and the cytosol, was used as a model ß-barrel IMP. Molecular dynamics simulations were used for comparison with the experimental results and to inform the parameters of the physical models describing the NR data. The detailed structure of the SAM is shown to depend on the density of the NTA chelating groups. The relative content of detergent and protein in surface-immobilized, detergent-stabilized VDAC is measured, while the reconstituted lipid bilayer is shown to be complete to within a few percent, using the known atomic structure of VDAC. Finally, excess lipid above the reconstituted bilayer, which is of consequence for more indirect structural and functional studies, is shown to be present.

Structural and functional insights into the role of BamD and BamE within the ß-barrel assembly machinery in Neisseria gonorrhoeae.

The ß-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Given its role in the production of OMPs for survival and pathogenesis, BAM represents an attractive target for the development of therapeutic interventions, including drugs and vaccines against multidrug-resistant bacteria such as Neisseria gonorrhoeae The first structure of BamA, the central component of BAM, was from N. gonorrhoeae, the etiological agent of the sexually transmitted disease gonorrhea. To aid in pharmaceutical targeting of BAM, we expanded our studies to BamD and BamE within BAM of this clinically relevant human pathogen. We found that the presence of BamD, but not BamE, is essential for gonococcal viability. However, BamE, but not BamD, was cell-surface-displayed under native conditions; however, in the absence of BamE, BamD indeed becomes surface-exposed. Loss of BamE altered cell envelope composition, leading to slower growth and an increase in both antibiotic susceptibility and formation of membrane vesicles containing greater amounts of vaccine antigens. Both BamD and BamE are expressed in diverse gonococcal isolates, under host-relevant conditions, and throughout different phases of growth. The solved structures of Neisseria BamD and BamE share overall folds with Escherichia coli proteins but contain differences that may be important for function. Together, these studies highlight that, although BAM is conserved across Gram-negative bacteria, structural and functional differences do exist across species, which may be leveraged in the development of species-specific therapeutics in the effort to combat multidrug resistance.