Modeling intermediates of BamA folding an outer membrane protein.

BamA, the core component of the ß-barrel assembly machinery complex, is an integral outer-membrane protein (OMP) in Gram-negative bacteria that catalyzes the folding and insertion of OMPs. A key feature of BamA relevant to its function is a lateral gate between its first and last ß-strands. Opening of this lateral gate is one of the first steps in the asymmetric-hybrid-barrel model of BamA function. In this study, multiple hybrid-barrel folding intermediates of BamA and a substrate OMP, EspP, were constructed and simulated to better understand the model's physical consequences. The hybrid-barrel intermediates consisted of the BamA ß-barrel and its POTRA5 domain and either one, two, three, four, five, or six ß-hairpins of EspP. The simulation results support an asymmetric-hybrid-barrel model in which the BamA N-terminal ß-strand forms stronger interactions with the substrate OMP than the C-terminal ß-strand. A consistent "B"-shaped conformation of the final folding intermediate was observed, and the shape of the substrate ß-barrel within the hybrid matched the shape of the fully folded substrate. Upon further investigation, inward-facing glycines were found at sharp bends within the hybrid and fully folded ß-barrels. Together, the data suggest an influence of sequence on shape of the substrate barrel throughout the OMP folding process and of the fully folded OMP.

Point Mutations in TbpA Abrogate Human Transferrin Binding in Neisseria gonorrhoeae.

TonB-dependent transporters (TDTs) are essential proteins for metal acquisition, an important step in the growth and pathogenesis of many pathogens, including Neisseria gonorrhoeae, the causative agent of gonorrhea. There is currently no available vaccine for gonorrhea; TDTs are being investigated as vaccine candidates because they are highly conserved and expressed in vivo. Transferrin binding protein A (TbpA) is an essential virulence factor in the initiation of experimental infection in human males and functions by acquiring iron upon binding to host transferrin (human transferrin [hTf]). The loop 3 helix (L3H) is a helix finger that inserts into the hTf C-lobe and is required for hTf binding and subsequent iron acquisition. This study identified and characterized the first TbpA single-point substitutions resulting in significantly decreased hTf binding and iron acquisition, suggesting that the helix structure is more important than charge for hTf binding and utilization. The tbpA D355P ΔtbpB and tbpA A356P ΔtbpB mutants demonstrated significantly reduced hTf binding and impaired iron uptake from Fe-loaded hTf; however, only the tbpA A356P ΔtbpB mutant was able to grow when hTf was the sole source of iron. The expression of tbpB was able to restore function in all tbpA mutants. These results implicate both D355 and A356 in the key binding, extraction, and uptake functions of gonococcal TbpA.

The assembly of ß-barrel membrane proteins by BAM and SAM.

Gram-negative bacteria, mitochondria, and chloroplasts all possess an outer membrane populated with a host of ß-barrel outer-membrane proteins (ßOMPs). These ßOMPs play crucial roles in maintaining viability of their hosts, and therefore, it is essential to understand the biogenesis of this class of membrane proteins. In recent years, significant structural and functional advancements have been made toward elucidating this process, which is mediated by the ß-barrel assembly machinery (BAM) in Gram-negative bacteria, and by the sorting and assembly machinery (SAM) in mitochondria. Structures of both BAM and SAM have now been reported, allowing a comparison and dissection of the two machineries, with other studies reporting on functional aspects of each. Together, these new insights provide compelling support for the proposed budding mechanism, where each nascent ßOMP forms a hybrid-barrel intermediate with BAM/SAM in route to its biogenesis into the membrane. Here, we will review these recent studies and highlight their contributions toward understanding ßOMP biogenesis in Gram-negative bacteria and in mitochondria. We will also weigh the evidence supporting each of the two leading mechanistic models for how BAM/SAM function, and offer an outlook on future studies within the field.

C-terminal kink formation is required for lateral gating in BamA.

In Gram-negative bacteria, the outer membrane contains primarily ß-barrel transmembrane proteins and lipoproteins. The insertion and assembly of ß-barrel outer-membrane proteins (OMPs) is mediated by the ß-barrel assembly machinery (BAM) complex, the core component of which is the 16-stranded transmembrane ß-barrel BamA. Recent studies have indicated a possible role played by the seam between the first and last ß-barrel strands of BamA in the OMP insertion process through lateral gating and a destabilized membrane region. In this study, we have determined the stability and dynamics of the lateral gate through over 12.5 µs of equilibrium simulations and 4 µs of free-energy calculations. From the equilibrium simulations, we have identified a persistent kink in the C-terminal strand and observed spontaneous lateral-gate separation in a mimic of the native bacterial outer membrane. Free-energy calculations of lateral gate opening revealed a significantly lower barrier to opening in the C-terminal kinked conformation; mutagenesis experiments confirm the relevance of C-terminal kinking to BamA structure and function.

Structural basis for substrate selection by the translocation and assembly module of the ß-barrel assembly machinery.

The assembly of proteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediated by the ß-barrel assembly machinery (BAM). Two crucial elements of that machinery are the core BAM complex and the translocation and assembly module (TAM), with each containing a member of the Omp85 superfamily of proteins: BamA in the BAM complex, TamA in the TAM. Here, we used the substrate protein FimD as a model to assess the selectivity of substrate interactions for the TAM relative to those of the BAM complex. A peptide scan revealed that TamA and BamA bind the ß-strands of FimD, and do so selectively. Chemical cross-linking and molecular dynamics are consistent with this interaction taking place between the first and last strand of the TamA barrel domain, providing the first experimental evidence of a lateral gate in TamA: a structural element implicated in membrane protein assembly. We suggest that the lateral gates in TamA and BamA provide different environments for substrates to engage, with the differences observed here beginning to address how the TAM can be more effective than the BAM complex in the folding of some substrate proteins.

Living on the edge: Simulations of bacterial outer-membrane proteins.

Gram-negative bacteria are distinguished in part by a second, outer membrane surrounding them. This membrane is distinct from others, possessing an outer leaflet composed not of typical phospholipids but rather large, highly charged molecules known as lipopolysaccharides. Therefore, modeling the structure and dynamics of proteins embedded in the outer membrane requires careful consideration of their native environment. In this review, we examine how simulations of such outer-membrane proteins have evolved over the last two decades, culminating most recently in detailed, highly accurate atomistic models of the outer membrane. We also draw attention to how the simulations have coupled with experiments to produce novel insights unattainable through a single approach. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

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. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation.

Structure Determination of Membrane Proteins Using X-Ray Crystallography.

Membrane proteins serve essential roles in all aspects of life and make up roughly one-third of all genomes from prokaryotes to eukaryotes. Their responsibilities include mediating cell signaling, nutrient import, waste export, cellular communication, trafficking, and immunity. For their critical role in many cellular processes, membrane proteins serve as targets for up to 50% of drugs currently on the market and remain primary targets in new therapeutics being developed. Despite their importance and abundance in nature, only ~1% of structures in the Protein Data Bank are of transmembrane proteins. This discrepancy can be directly attributed to the biochemical properties of membrane proteins and the difficulty in producing sufficient yields for structural studies or the difficulty in growing well-ordered crystals. Here, we present methods from our work that outline our general pipeline from cloning to structure determination of membrane proteins, with a focus on using X-ray crystallography, which still yields ~90% of all structures being deposited into the Protein Data Bank.

Plasticity within the barrel domain of BamA mediates a hybrid-barrel mechanism by BAM.

In Gram-negative bacteria, the biogenesis of ß-barrel outer membrane proteins is mediated by the ß-barrel assembly machinery (BAM). The mechanism employed by BAM is complex and so far- incompletely understood. Here, we report the structures of BAM in nanodiscs, prepared using polar lipids and native membranes, where we observe an outward-open state. Mutations in the barrel domain of BamA reveal that plasticity in BAM is essential, particularly along the lateral seam of the barrel domain, which is further supported by molecular dynamics simulations that show conformational dynamics in BAM are modulated by the accessory proteins. We also report the structure of BAM in complex with EspP, which reveals an early folding intermediate where EspP threads from the underside of BAM and incorporates into the barrel domain of BamA, supporting a hybrid-barrel budding mechanism in which the substrate is folded into the membrane sequentially rather than as a single unit.

Presence of substrate aids lateral gate separation in LptD.

Lipopolysaccharides (LPS) provide the outer membrane (OM) of Gram-negative bacteria with a strong protective barrier. The periplasm-spanning Lpt machinery is responsible for the transport of LPS molecules across the periplasm, culminating in insertion by the outer-membrane proteins LptD and LptE. In order to elucidate the mechanisms of LPS insertion by LptDE, we performed over 14 microseconds of equilibrium molecular dynamics simulations. Bilayer-dependent differences in the fluctuations and secondary structure of LptD's extracellular loops are observed for a pure DMPE membrane vs. a model of the OM. Furthermore, LptD's periplasmic N-terminal domain is highly dynamic, which may help to maintain the integrity of the periplasm-spanning complex amidst relative motion of the inner-membrane and outer-membrane anchored domains. In addition, our simulations demonstrate that binding of LPS substrate activates a switching between the associated and dissociated states of two lumenal loops at the interface between the ß-barrel and the N-terminal domain as well as LptD's lateral gate on the microsecond timescale, neither of which is observed for the apo state. Placement of a substrate LPS molecule also causes an increase in the average separation of the LptD lateral gate strands and a lowering of the energetic barrier to lateral gate opening.

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.

BamA is required for autotransporter secretion.

BACKGROUND: In Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the "passenger" domain) and a ß-barrel that aids its export. While it is known that the folding and insertion of the ß-barrel domain utilize the ß-barrel assembly machinery (BAM) complex, how the passenger domain is secreted and folded across the membrane remains to be determined. The hairpin model states that passenger domain secretion occurs independently through the fully-formed and membrane-inserted ß-barrel domain via a hairpin folding intermediate. In contrast, the BamA-assisted model states that the passenger domain is secreted through a hybrid of BamA, the essential subunit of the BAM complex, and the ß-barrel domain of the autotransporter. METHODS: To ascertain the models' plausibility, we have used molecular dynamics to simulate passenger domain secretion for two autotransporters, EspP and YadA. RESULTS: We observed that each protein's ß-barrel is unable to accommodate the secreting passenger domain in a hairpin configuration without major structural distortions. Additionally, the force required for secretion through EspP's ß-barrel is more than that through the BamA ß-barrel. CONCLUSIONS: Secretion of autotransporters most likely occurs through an incompletely formed ß-barrel domain of the autotransporter in conjunction with BamA. GENERAL SIGNIFICANCE: Secretion of virulence factors is a process used by practically all pathogenic Gram-negative bacteria. Understanding this process is a necessary step towards limiting their infectious capacity.

Molecular Insight into TdfH-Mediated Zinc Piracy from Human Calprotectin by Neisseria gonorrhoeae.

Neisseria gonorrhoeae, responsible for the sexually transmitted infection gonorrhea, is an obligate human pathogen exquisitely adapted for survival on mucosal surfaces of humans. This host-pathogen relationship has resulted in evolution by N. gonorrhoeae of pathways that enable the use of host metalloproteins as required nutrients through the deployment of outer membrane-bound TonB-dependent transporters (TdTs). Recently, a TdT called TdfH was implicated in binding to calprotectin (CP) and in removal of the bound zinc (Zn), enabling gonococcal growth. TdfH is highly conserved among the pathogenic Neisseria species, making it a potentially promising candidate for inclusion into a gonococcal vaccine. Currently, the nature and specificity of the TdfH-CP interaction have not been determined. In this study, we found that TdfH specifically interacted with human calprotectin (hCP) and that growth of the gonococcus was supported in a TdfH-dependent manner only when hCP was available as a sole zinc source and not when mouse CP was provided. The binding interactions between TdfH and hCP were assessed using isothermal titration calorimetry where we observed a multistate model having both high-affinity and low-affinity sites of interaction. hCP has two Zn binding sites, and gonococcal growth assays using hCP mutants deficient in one or both of the Zn binding sites revealed that TdfH exhibited a site preference during Zn piracy and utilization. This report provides the first insights into the molecular mechanism of Zn piracy by neisserial TdfH and further highlights the obligate human nature of N. gonorrhoeae and the high-affinity interactions occurring between TdTs and their human ligands during pathogenesis.IMPORTANCE The dramatic rise in antimicrobial resistance among Neisseria gonorrhoeae isolates over the last few decades, paired with dwindling treatment options and the lack of a protective vaccine, has prompted increased interest in identifying new bacterial targets for the treatment and, ideally, prevention of gonococcal disease. TonB-dependent transporters are a conserved set of proteins that serve crucial functions for bacterial survival within the host. In this study, binding between the gonococcal transporter, TdfH, and calprotectin was determined to be of high affinity and host restricted. The current study identified a preferential TdfH interaction at the calprotectin dimer interface. An antigonococcal therapeutic could potentially block this site on calprotectin, interrupting Zn uptake by N. gonorrhoeae and thereby prohibiting continued bacterial growth. We describe protein-protein interactions between TdfH and calprotectin, and our findings provide the building blocks for future therapeutic or prophylactic targets.

Accelerating Membrane Simulations with Hydrogen Mass Repartitioning.

The time step of atomistic molecular dynamics (MD) simulations is determined by the fastest motions in the system and is typically limited to 2 fs. An increasingly popular approach is to increase the mass of the hydrogen atoms to ∼3 amu and decrease the mass of the parent atom by an equivalent amount. This approach, known as hydrogen-mass repartitioning (HMR), permits time steps up to 4 fs with reasonable simulation stability. While HMR has been applied in many published studies to date, it has not been extensively tested for membrane-containing systems. Here, we compare the results of simulations of a variety of membranes and membrane-protein systems run using a 2 fs time step and a 4 fs time step with HMR. For pure membrane systems, we find almost no difference in structural properties, such as area-per-lipid, electron density profiles, and order parameters, although there are differences in kinetic properties such as the diffusion constant. Conductance through a porin in an applied field, partitioning of a small peptide, hydrogen-bond dynamics, and membrane mixing show very little dependence on HMR and the time step. We also tested a 9 Å cutoff as compared to the standard CHARMM cutoff of 12 Å, finding significant deviations in many properties tested. We conclude that HMR is a valid approach for membrane systems, but a 9 Å cutoff is not.

A novel human IL2RB mutation results in T and NK cell-driven immune dysregulation.

The pleiotropic actions of interleukin-2 (IL-2) are essential for regulation of immune responses and maintenance of immune tolerance. The IL-2 receptor (IL-2R) is composed of IL-2Rα, IL-2Rß, and IL-2Rγ subunits, with defects in IL-2Rα and IL-2Rγ and their downstream signaling effectors resulting in known primary immunodeficiency disorders. Here, we report the first human defect in IL-2Rß, occurring in two infant siblings with a homozygous IL2RB mutation in the WSXWS motif, manifesting as multisystem autoimmunity and susceptibility to CMV infection. The hypomorphic mutation results in diminished IL-2Rß surface expression and dysregulated IL-2/15 signaling, with an anticipated reduction in regulatory T cells. However, in contrast to the IL-2Rß-/- animal model, which lacks NK cells, these siblings demonstrate an expansion of NK cells, particularly the CD56bright subset, and a lack of terminally differentiated NK cells. Thus, the early-onset autoimmunity and immunodeficiency are linked to functional deficits arising from altered IL-2Rß expression and signaling in T and NK cells.

Accelerating the use of molecular modeling in the high school classroom with VMD Lite.

It is often difficult for students to develop an intuition about molecular processes, which occur in a realm far different from day-to-day life. For example, thermal fluctuations take on hurricane-like proportions at the molecular scale. Students need a way to visualize realistic depictions of molecular processes to appreciate them. To this end, we have developed a simplified graphical interface to the widely used molecular visualization and analysis tool Visual Molecular Dynamics (VMD) called VMD lite. We demonstrate the use of VMD lite through a module on diffusion and the hydrophobic effect as they relate to membrane formation. Trajectories from molecular dynamics simulations, which students can interact with freely, illustrate the dynamical behavior of lipid molecules and water. VMD lite was tested by ∼70 students with overall positive reception. Remaining deficiencies in conceptual understanding were noted, however, and the module has been revised in response.

Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens.

Incorporation of lipopolysaccharide (LPS) into the outer membrane of Gram-negative bacteria is essential for viability, and is accomplished by a two-protein complex called LptDE. We solved crystal structures of the core LptDE complexes from Yersinia pestis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and a full-length structure of the K. pneumoniae LptDE complex. Our structures adopt the same plug and 26-strand ß-barrel architecture found recently for the Shigella flexneri and Salmonella typhimurium LptDE structures, illustrating a conserved fold across the family. A comparison of the only two full-length structures, SfLptDE and our KpLptDE, reveals a 21° rotation of the LptD N-terminal domain that may impart flexibility on the trans-envelope LptCAD scaffold. Utilizing mutagenesis coupled to an in vivo functional assay and molecular dynamics simulations, we demonstrate the critical role of Pro231 and Pro246 in the function of the LptD lateral gate that allows partitioning of LPS into the outer membrane.