Vassiliy N. Bavro

Assembly and Channel Opening in a Bacterial Drug Efflux Machine.

Summary Drugs and certain proteins are transported across the membranes of Gram-negative bacteria by energy-activated pumps. The outer membrane component of these pumps is a channel that opens from a sealed resting state during the transport process. We describe two crystal structures of the Escherichia coli outer membrane protein TolC in its partially open state. Opening is accompanied by the exposure of three shallow intraprotomer grooves in the TolC trimer, where our mutagenesis data identify a contact point with the periplasmic component of a drug efflux pump, AcrA. We suggest that the assembly of multidrug efflux pumps is accompanied by induced fit of TolC driven mainly by accommodation of the periplasmic component.

Structural basis of dynamic glycine receptor clustering by gephyrin

Gephyrin is a bi‐functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full‐length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C‐terminal region (gephyrin‐E), which binds a GlyR β‐subunit‐derived peptide with high and low affinity. The crystal structure of the tetra‐domain gephyrin‐E in complex with the β‐peptide bound to domain IV indicates how membrane‐embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full‐length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full‐length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity.

Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.

A model of a transmembrane drug-efflux pump from Gram-negative bacteria.

In Gram‐negative bacteria, drug resistance is due in part to the activity of transmembrane efflux‐pumps, which are composed of three types of proteins. A representative pump from Escherichia coli is an assembly of the trimeric outer‐membrane protein TolC, which is an allosteric channel, the trimeric inner‐membrane proton‐antiporter AcrB, and the periplasmic protein, AcrA. The pump displaces drugs vectorially from the bacterium using proton electrochemical force. Crystal structures are available for TolC and AcrB from E. coli, and for the AcrA homologue MexA from Pseudomonas aeruginosa. Based on homology modelling and molecular docking, we show how AcrA, AcrB and TolC might assemble to form a tripartite pump, and how allostery may occur during transport.

Ion Mobility Mass Spectrometry of Two Tetrameric Membrane Protein Complexes Reveals Compact Structures and Differences in Stability and Packing

Here we examined the gas-phase structures of two tetrameric membrane protein complexes by ion mobility mass spectrometry. The collision cross sections measured for the ion channel are in accord with a compact configuration of subunits, suggesting that the native-like structure can be preserved under the harsh activation conditions required to release it from the detergent micelle into the gas phase. We also found that the quaternary structure of the transporter, which has fewer transmembrane subunits than the ion channel, is less stable once stripped of detergents and bulk water. These results highlight the potential of ion mobility mass spectrometry for characterizing the overall topologies of membrane protein complexes and the structural changes associated with nucleotide, lipid, and drug binding.

AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity

Significance Genome sequencing of a multidrug-resistant clinical isolate of Salmonella Typhimurium from a patient that failed ciprofloxacin therapy revealed a mutation in the efflux pump gene, acrB. Computational modelling revealed that the G288D substitution changed the binding of drugs to the distal binding pocket of AcrB. The mutation was recreated in an unrelated Salmonella strain and also in Escherichia coli; in both species the efflux of ciprofloxacin was increased by the mutation, explaining its resistant phenotype. This is the first time a substitution within an efflux pump protein has been shown to cause drug resistance. Importantly, the finding that one amino acid change can cause resistance to some drugs, but susceptibility to others, informs those developing new antibiotics. The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies.

MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

Assembly and transport mechanism of tripartite drug efflux systems

Multidrug efflux (MDR) pumps remove a variety of compounds from the cell into the external environment. There are five different classes of MDR pumps in bacteria, and quite often a single bacterial species expresses multiple classes of pumps. Although under normal circumstances MDR pumps confer low-level intrinsic resistance to drugs, the presence of drugs and mutations in regulatory genes lead to high level expression of MDR pumps that can pose problems with therapeutic treatments. This review focuses on the resistance nodulation cell division (RND)-class of MDR pumps that assemble from three proteins. Significant recent advancement in structural aspects of the three pump components has shed new light on the mechanism by which the tripartite efflux pumps extrude drugs. This new information will be critical in developing inhibitors against MDR pumps to improve the potency of prescribed drugs.

Crystal structure of the GABAA‐receptor‐associated protein, GABARAP

The GABAA‐receptor‐associated protein (GABARAP) is a member of a growing family of intracellular membrane trafficking and/or fusion proteins and has been implicated in plasma membrane targeting and/or recycling of GABAA receptors. GABARAP is localized on intracellular membranes such as the trans‐Golgi network, binds to the γ 2 subunit of GABAA receptors and interacts with microtubules and the N‐ethylmaleimide‐sensitive factor. We report the X‐ray crystal structure of mammalian GABARAP at 2.0 Å resolution. GABARAP consists of an N‐terminal basic helical region, which has been implicated in tubulin binding, and a core structure with a conserved ubiquitin‐like fold. Consistent with the high extent of sequence conservation among GABARAP homologues from plants to mammals, one face of the core structure is absolutely conserved while the opposite face shows considerable divergence. These features are in agreement with the conserved surface mediating protein–protein interactions shared by all members of the family, whereas the non‐conserved surface region may play specific roles, such as docking to particular membrane receptors.

Conformational Changes During the Gating of a Potassium Channel Revealed by Structural Mass Spectrometry

Potassium channels are dynamic proteins that undergo large conformational changes to regulate the flow of K(+) ions across the cell membrane. Understanding the gating mechanism of these channels therefore requires methods for probing channel structure in both their open and closed conformations. Radiolytic footprinting is used to study the gating mechanism of the inwardly-rectifying potassium channel KirBac3.1. The purified protein stabilized in either open or closed conformations was exposed to focused synchrotron X-ray beams on millisecond timescales to modify solvent accessible amino acid side chains. These modifications were identified and quantified using high-resolution mass spectrometry. The differences observed between the closed and open states were then used to reveal local conformational changes that occur during channel gating. The results provide support for a proposed gating mechanism of the Kir channel and demonstrate a method of probing the dynamic gating mechanism of other integral membrane proteins and ion channels.