OccK channels from Pseudomonas aeruginosa exhibit diverse single-channel electrical signatures but conserved anion selectivity

Pseudomonas aeruginosa is a Gram-negative bacterium that utilizes substrate-specific outer membrane (OM) proteins for the uptake of small, water-soluble nutrients employed in the growth and function of the cell. In this paper, we present for the first time a comprehensive single-channel examination of seven members of the OM carboxylate channel K (OccK) subfamily. Recent biochemical, functional, and structural characterization of the OccK proteins revealed their common features, such as a closely related, monomeric, 18-stranded β-barrel conformation with a kidney-shaped transmembrane pore and the presence of a basic ladder within the channel lumen. Here, we report that the OccK proteins exhibited fairly distinct unitary conductance values, in a much broader range than previously expected, which includes low (~40-100 pS) and medium (~100-380 pS) conductance. These proteins showed diverse single-channel dynamics of current gating transitions, revealing one-open substate (OccK3), two-open substate (OccK4-OccK6), and three-open substate (OccK1, OccK2, and OccK7) kinetics with functionally distinct conformations. Interestingly, we discovered that anion selectivity is a conserved trait among the members of the OccK subfamily, confirming the presence of a net pool of positively charged residues within their central constriction. Moreover, these results are in accord with an increased specificity and selectivity of these protein channels for negatively charged, carboxylate-containing substrates. Our findings might ignite future functional examinations and full atomistic computational studies for unraveling a mechanistic understanding of the passage of small molecules across the lumen of substrate-specific, β-barrel OM proteins.     

Analysis of gating transitions among the three major open states of the OpdK channel

OpdK is an outer membrane protein of the pathogenic bacterium Pseudomonas aeruginosa. The recent crystal structure of this protein revealed a monomeric, 18-stranded β-barrel with a kidney-shaped pore, whose constriction features a diameter of 8 ?. Using systematic single-channel electrical recordings of this protein pore reconstituted into planar lipid bilayers under a broad range of ion concentrations, we were able to probe its discrete gating kinetics involving three major and functionally distinct conformations, in which a dominant open substate O(2) is accompanied by less thermodynamically stable substates O(1) and O(3). Single-channel electrical data enabled us to determine the alterations in the energetics and kinetics of the OpdK protein when experimental conditions were changed. In the future, such a semiquantitative analysis might provide a better understanding on the dynamics of current fluctuations of other β-barrel membrane protein channels.     

A Tag at the Carboxy Terminus Prevents Membrane Integration of VDAC1 in Mammalian Mitochondria

β-Barrel proteins are found in the outer membranes of bacteria, chloroplasts and mitochondria. The evolutionary conserved sorting and assembly machinery (SAM complex) assembles mitochondrial β-barrel proteins, such as voltage-dependent anion-selective channel 1 (VDAC1), into complexes in the outer membrane by recognizing a sorting β-signal in the carboxy-terminal part of the protein. Here we show that in mammalian mitochondria, masking of the C-terminus of β-barrel proteins by a tag leads to accumulation of soluble misassembled protein in the intermembrane space, which causes mitochondrial fragmentation and loss of membrane potential. A similar phenotype is observed if the β-signal is shortened, removed or when the conserved hydrophobic residues in the β-signal are mutated. The length of the tag at the C-terminus is critical for the assembly of VDAC1, as well as the amino acid residues at positions 130, 222, 225 and 251 of the protein. We propose that if the recognition of the β-signal or the folding of the β-barrel proteins is inhibited, the nonassembled protein will accumulate in the intermembrane space, aggregate and damage mitochondria. This effect offers easy tools for studying the requirements for the membrane assembly of β-barrel proteins, but also advises caution when interpreting the outcome of the β-barrel protein overexpression experiments.     

Modifying the pH sensitivity of OmpG nanopore for improved detection at acidic pH

The outer membrane protein G (OmpG) nanopore is a monomeric β-barrel channel consisting of seven flexible extracellular loops. Its most flexible loop, loop 6, can be used to host high-affinity binding ligands for the capture of protein analytes, which induces characteristic current patterns for protein identification. At acidic pH, the ability of OmpG to detect protein analytes is hampered by its tendency toward the closed state, which renders the nanopore unable to reveal current signal changes induced by bound analytes. In this work, critical residues that control the pH-dependent gating of loop 6 were identified, and an OmpG nanopore that can stay predominantly open at a broad range of pHs was created by mutating these pH-sensitive residues. A short single-stranded DNA was chemically tethered to the pH-insensitive OmpG to demonstrate the utility of the OmpG nanopore for sensing complementary DNA and a DNA binding protein at an acidic pH.     

Gating and conduction of nano-channel forming proteins: a computational approach

Monitoring conformational changes in ion channels is essential to understand their gating mechanism. Here, we explore the structural dynamics of four outer membrane proteins with different structures and functions in the slowest nonzero modes of vibration. Normal mode analysis was performed on the modified elastic network model of channel in the membrane. According to our results, when membrane proteins were analyzed in the dominant mode, the composed pores, TolC and α-hemolysin showed large motions at the intramembrane β-barrel region while, in other porins, OmpA and OmpF, largest motions observed in the region of external flexible loops. A criterion based on equipartition theorem was used to measure the possible amplitude of vibration in channel forming proteins. The current approach complements theoretical and experimental techniques including HOLE, Molecular Dynamics (MD), and voltage clamp used to address the channel’s structure and dynamics and provides the means to conduct a theoretical simultaneous study of the structure and function of the channel.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:3     

Redesign of a plugged beta-barrel membrane protein

The redesign of biological nanopores is focused on bacterial outer membrane proteins and pore-forming toxins, because their robust β-barrel structure makes them the best choice for developing stochastic biosensing elements. Using membrane protein engineering and single-channel electrical recordings, we explored the ferric hydroxamate uptake component A (FhuA), a monomeric 22-stranded β-barrel protein from the outer membrane of Escherichia coli. FhuA has a luminal cross-section of 3.1 × 4.4 nm and is filled by a globular N-terminal cork domain. Various redesigned FhuA proteins were investigated, including single, double, and multiple deletions of the large extracellular loops and the cork domain. We identified four large extracellular loops that partially occlude the lumen when the cork domain is removed. The newly engineered protein, FhuAΔC/Δ4L, was the result of a removal of almost one-third of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. This extensive protein engineering encompassed the entire cork domain and four extracellular loops. Remarkably, FhuAΔC/Δ4L forms a functional open pore in planar lipid bilayers, with a measured unitary conductance of ～4.8 nanosiemens, which is much greater than the values recorded previously with other engineered FhuA protein channels. There are numerous advantages and prospects of using such an engineered outer membrane protein not only in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating, but also in more applicative areas of stochastic single-molecule sensing of proteins and nucleic acids.     

The signal distinguishing between targeting of outer membrane β-barrel protein to plastids and mitochondria in plants

The proteome of the outer membrane of mitochondria and chloroplasts consists of membrane proteins anchored by α-helical or β-sheet elements. While proteins with α-helical transmembrane domains are present in all cellular membranes, proteins with β-barrel structure are specific for these two membranes. The organellar β-barrel proteins are encoded in the nuclear genome and thus, have to be targeted to the outer organellar membrane where they are recognized by surface exposed translocation complexes. In the last years, the signals that ensure proper targeting of these proteins have been investigated as essential base for an understanding of the regulation of cellular protein distribution. However, the organellar β-barrel proteins are unique as most of them do not contain a typical targeting information in form of an N-terminal cleavable targeting signal. Recently, it was discovered that targeting and surface recognition of mitochondrial β-barrel proteins in yeast, humans and plants depends on the hydrophobicity of the last β-hairpin of the β-barrel. However, we demonstrate that the hydrophobicity is not sufficient for the discrimination of targeting to chloroplasts or mitochondria. By domain swapping between mitochondrial and chloroplast targeted β-barrel proteins atVDAC1 and psOEP24 we demonstrate that the presence of a hydrophilic amino acid at the C-terminus of the penultimate β-strand is required for mitochondrial targeting. A mutation of the chloroplast β-barrel protein psOEP24 which mimics such profile is efficiently targeted to mitochondria. Thus, we present the properties of the signal for mitochondrial targeting of β-barrel proteins in plants.     

pH-Induced Conformational Change of the β-Barrel-Forming Protein OmpG Reconstituted into Native E. coli Lipids

A gating mechanism of the β-barrel-forming outer membrane protein G (OmpG) from Escherichia coli was recently presented. The mechanism was based on X-ray structures revealed from crystals grown from solubilized OmpG at both neutral pH and acidic pH. To investigate whether these conformations represent the naturally occurring gating mechanism, we reconstituted OmpG in native E. coli lipids and applied high-resolution atomic force microscopy. The reconstituted OmpG molecules assembled into both monomers and dimers. Single monomeric and dimeric OmpG molecules showed open channel entrances at pH 7.5 and at room temperature. The extracellular loops connecting the β-strands that form the transmembrane β-barrel pore exhibited elevated structural flexibility. Upon lowering the pH to 5.0, the conformation of OmpG molecules changed to close the extracellular entrance of their channel. It appears that one or more of the extracellular loops collapsed onto the channel entrance. This conformational change was fully reversible. Our data confirm that the previously reported gating mechanism of OmpG occurs at physiological conditions in E. coli lipid membranes.     

Crystal Structures of CusC Review Conformational Changes Accompanying Folding and Transmembrane Channel Formation

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the RND (resistance–nodulation–cell division) family to expel diverse toxic compounds from the cell. These complexes span both the inner and outer membranes of the bacterium via an α-helical, inner membrane transporter; a periplasmic membrane fusion protein; and a β-barrel, outer membrane channel. One such efflux system, CusCBA, is responsible for extruding biocidal Cu(I) and Ag(I) ions. To remove these toxic ions, the CusC outer membrane channel must form a β-barrel structural domain, which creates a pore and spans the entire outer membrane. We here report the crystal structures of wild-type CusC, as well as two CusC mutants, suggesting that the first N-terminal cysteine residue plays an important role in protein–membrane interactions and is critical for the insertion of this channel protein into the outer membrane. These structures provide insight into the mechanisms on CusC folding and transmembrane channel formation. It is found that the interactions between CusC and membrane may be crucial for controlling the opening and closing of this β-barrel, outer membrane channel.     

Structural and functional characterization of a synthetically modified OmpG

Chemical modification of ion channels has recently attracted attention due to their potential use in stochastic sensing and neurobiology. Among the available channel templates stable β-barrel proteins have shown their potential for large scale chemical modifications due to their wide pore lumen. Ion-channel hybrids using the outer membrane protein OmpG were generated by S-alkylation with a synthetic modulator and functionally as well as structurally characterized. The dansyl moiety of the used modulator resulted in partial blockage of current though the OmpG channel with its gating characteristics mainly unaffected. The crystal structure of an OmpG–dansyl hybrid at 2.4 Å resolution correlates this finding by showing that the modulator lines the inner walling of the OmpG pore. These results underline the suitability of OmpG as a structural base for the construction of stochastic sensors.     

The sodium channel activator Brevetoxin-3 uncovers a multiplicity of different open states of the cardiac sodium channel.

The interaction of Brevetoxin 3 (Pbtx-3), a sodium channel activator, with the cardiac sodium channel was studied at the single channel level. It was found that Pbtx-3 (20 microM) shifted steady-state activation to negative potentials, without major effects on the time course of macroscopic activation or macroscopic currents decay, as calculated from averaged single-channel records. Single-channel open times were found to be prolonged. Under the influence of the toxin, sodium channel openings could be observed frequently even at maintained depolarisation. These openings occurred to at least nine different subconductance levels of the open state with smaller conductivities than the maximal one and differed in their open times. Current amplitudes of these open substates were found to cluster around certain amplitude values. Appearance of substates at maintained depolarisation was dependent on the transmembrane potential (Em): Substates with smaller conductivity appeared more frequently at lower Em values whereas at higher Em values substates with higher conductivity values dominated. Furthermore, it was demonstrated that appearance of substates did not result from incomplete recovery from inactivation. From these observations it was concluded that the open substates observed correspond to different conformational states of the channel's activation gates. Under physiological conditions, when the sodium channel opens directly from its closed state these 'incomplete'-open states of the cardiac sodium channel are obscured by fast gating transitions between the corresponding, electrically silent, preopen states. Thus, Pbtx-3 acts mainly via stabilisation of the channel's preopen and different open states. A classification of sodium channel modifiers, based on their interaction with different conformational states of the channel is suggested.     

Mitochondria can recognize and assemble fragments of a beta-barrel structure

β-barrel proteins are found in the outer membranes of eukaryotic organelles of endosymbiotic origin as well as in the outer membrane of Gram-negative bacteria. Precursors of mitochondrial β-barrel proteins are synthesized in the cytosol and have to be targeted to the organelle. Currently, the signal that assures their specific targeting to mitochondria is poorly defined. To characterize the structural features needed for specific mitochondrial targeting and to test whether a full β-barrel structure is required, we expressed in yeast cells the β-barrel domain of the trimeric autotransporter Yersinia adhesin A (YadA). Trimeric autotransporters are found only in prokaryotes, where they are anchored to the outer membrane by a single 12-stranded β-barrel structure to which each monomer is contributing four β-strands. Importantly, we found that YadA is solely localized to the mitochondrial outer membrane, where it exists in a native trimeric conformation. These findings demonstrate that, rather than a linear sequence or a complete β-barrel structure, four β-strands are sufficient for the mitochondria to recognize and assemble a β-barrel protein. Remarkably, the evolutionary origin of mitochondria from bacteria enables them to import and assemble even proteins belonging to a class that is absent in eukaryotes.     

The structural biology of β-barrel membrane proteins: a summary of recent reports

The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts all contain transmembrane β-barrel proteins. These β-barrel proteins serve essential functions in cargo transport and signaling and are also vital for membrane biogenesis. They have also been adapted to perform a diverse set of important cellular functions including acting as porins, transporters, enzymes, virulence factors and receptors. Recent structures of transmembrane β-barrels include that of a full length autotransporter (EstA), a bacterial heme transporter complex (HasR), a bacterial porin in complex with several ligands (PorB), and the mitochondrial voltage-dependent anion channel (VDAC) from both mouse and human. These represent only a few of the interesting structures of β-barrel membrane proteins recently elucidated. However, they demonstrate many of the advancements made within the field of transmembrane protein structure in the past few years.     

Structural biology of membrane-intrinsic β-barrel enzymes: Sentinels of the bacterial outer membrane

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel β-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three β-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipid∷lipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane β-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two β-barrel enzymes of unknown structure; namely, the Salmonella enterica 3′-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O₂ to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how β-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger β-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.     

Biogenesis of β-barrel integral proteins of bacterial outer membrane

Gram-negative bacteria are enveloped by two membranes, the inner (cytoplasmic) (CM) and the outer (OM). The majority of integral outer membrane proteins are arranged in β-barrels of cylindrical shape composed of amphipathic antiparallel β-strands. In bacteria, β-barrel proteins function as water-filled pores, active transporters, enzymes, receptors, and structural proteins. Proteins of bacterial OM are synthesized in the cytoplasm as unfolded polypeptides with an N-terminal sequence that marks them for transport across the CM. Precursors of membrane proteins move through the aqueous medium of the cytosol and periplasm under the protection of chaperones (SecB, Skp, SurA, and DegP), then cross the CM via the Sec system composed of a polypeptide-conducting channel (SecYEG) and ATPase (SecA), the latter providing the energy for the translocation of the pre-protein. Pre-protein folding and incorporation in the OM require the participation of the Bam-complex, probably without the use of energy. This review summarizes current data on the biogenesis of the β-barrel proteins of bacterial OM. Data on the structure of the proteins included in the multicomponent system for delivery of the OM proteins to their destination in the cell and on their complexes with partners, including pre-proteins, are pre-sented. Molecular models constructed on the basis of structural, genetic, and biochemical studies that describe the mechanisms of β-barrel protein assembly by this molecular transport machinery are also considered.     

Outer membrane β-barrel structure prediction through the lens of AlphaFold2

Most proteins found in the outer membrane of gram-negative bacteria share a common domain: the transmembrane β-barrel. These outer membrane β-barrels (OMBBs) occur in multiple sizes and different families with a wide range of functions evolved independently by amplification from a pool of homologous ancestral ββ-hairpins. This is part of the reason why predicting their three-dimensional (3D) structure, especially by homology modeling, is a major challenge. Recently, DeepMind's AlphaFold v2 (AF2) became the first structure prediction method to reach close-to-experimental atomic accuracy in CASP even for difficult targets. However, membrane proteins, especially OMBBs, were not abundant during their training, raising the question of how accurate the predictions are for these families. In this study, we assessed the performance of AF2 in the prediction of OMBBs and OMBB-like folds of various topologies using an in-house-developed tool for the analysis of OMBB 3D structures, and barrOs. In agreement with previous studies on other membrane protein classes, our results indicate that AF2 predicts transmembrane β-barrel structures at high accuracy independently of the use of templates, even for novel topologies absent from the training set. These results provide confidence on the models generated by AF2 and open the door to the structural elucidation of novel transmembrane β-barrel topologies identified in high-throughput OMBB annotation studies or designed de novo.     

Substrate-dependent arrangements of the subunits of the BAM complex determined by neutron reflectometry

In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex catalyses the assembly of β-barrel proteins into the outer membrane, and is composed of five subunits: BamA, BamB, BamC, BamD and BamE. Once assembled, - β-barrel proteins can be involved in various functions including uptake of nutrients, export of toxins and mediating host-pathogen interactions, but the precise mechanism by which these ubiquitous and often essential β-barrel proteins are assembled is yet to be established. In order to determine the relative positions of BAM subunits in the membrane environment we reconstituted each subunit into a biomimetic membrane, characterizing their interaction and structural changes by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and neutron reflectometry. Our results suggested that the binding of BamE, or a BamDE dimer, to BamA induced conformational changes in the polypeptide transported-associated (POTRA) domains of BamA, but that BamB or BamD alone did not promote any such changes. As monitored by neutron reflectometry, addition of an unfolded substrate protein extended the length of POTRA domains further away from the membrane interface as part of the mechanism whereby the substrate protein was folded into the membrane.     

Pattern of amino acid substitutions in transmembrane domains of β-barrel membrane proteins for detecting remote homologs in bacteria and mitochondria

β-barrel membrane proteins play an important role in controlling the exchange and transport of ions and organic molecules across bacterial and mitochondrial outer membranes. They are also major regulators of apoptosis and are important determinants of bacterial virulence. In contrast to β-helical membrane proteins, their evolutionary pattern of residue substitutions has not been quantified, and there are no scoring matrices appropriate for their detection through sequence alignment. Using a Bayesian Monte Carlo estimator, we have calculated the instantaneous substitution rates of transmembrane domains of bacterial β-barrel membrane proteins. The scoring matrices constructed from the estimated rates, called bbTM for β-barrel Transmembrane Matrices, improve significantly the sensitivity in detecting homologs of β-barrel membrane proteins, while avoiding erroneous selection of both soluble proteins and other membrane proteins of similar composition. The estimated evolutionary patterns are general and can detect β-barrel membrane proteins very remote from those used for substitution rate estimation. Furthermore, despite the separation of 2-3 billion years since the proto-mitochondrion entered the proto-eukaryotic cell, mitochondria outer membrane proteins in eukaryotes can also be detected accurately using these scoring matrices derived from bacteria. This is consistent with the suggestion that there is no eukaryote-specific signals for translocation. With these matrices, remote homologs of β-barrel membrane proteins with known structures can be reliably detected at genome scale, allowing construction of high quality structural models of their transmembrane domains, at the rate of 131 structures per template protein. The scoring matrices will be useful for identification, classification, and functional inference of membrane proteins from genome and metagenome sequencing projects. The estimated substitution pattern will also help to identify key elements important for the structural and functional integrity of β-barrel membrane proteins, and will aid in the design of mutagenesis studies.     

Voltage-activated complexation of α-synuclein with three diverse β-barrel channels: VDAC,MspA, and α-hemolysin

Voltage-activated complexation is the process by which a transmembrane potential drives complex formation between a membrane-embedded channel and a soluble or membrane-peripheral target protein. Metabolite and calcium flux across the mitochondrial outer membrane was shown to be regulated by voltage-activated complexation of the voltage-dependent anion channel (VDAC) and either dimeric tubulin or α-synuclein (αSyn). However, the roles played by VDAC's characteristic attributes—its anion selectivity and voltage gating behavior—have remained unclear. Here, we compare in vitro measurements of voltage-activated complexation of αSyn with three well-characterized β-barrel channels—VDAC, MspA, and α-hemolysin—that differ widely in their organism of origin, structure, geometry, charge density distribution, and voltage gating behavior. The voltage dependences of the complexation dynamics for the different channels are observed to differ quantitatively but have similar qualitative features. In each case, energy landscape modeling describes the complexation dynamics in a manner consistent with the known properties of the individual channels, while voltage gating does not appear to play a role. The reaction free energy landscapes thus calculated reveal a non-trivial dependence of the αSyn/channel complex stability on the surface density of αSyn.