Publications by authors named "Vassiliy N Bavro"

40 Publications

Interchangeability of periplasmic adaptor proteins AcrA and AcrE in forming functional efflux pumps with AcrD in Salmonella enterica serovar Typhimurium.

J Antimicrob Chemother 2021 Jul 19. Epub 2021 Jul 19.

Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

Background: Resistance-nodulation-division (RND) efflux pumps are important mediators of antibiotic resistance. RND pumps, including the principal multidrug efflux pump AcrAB-TolC in Salmonella, are tripartite systems with an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). We previously identified the residues required for binding between the PAP AcrA and the RND transporter AcrB and have demonstrated that PAPs can function with non-cognate transporters. AcrE and AcrD/AcrF are homologues of AcrA and AcrB, respectively. Here, we show that AcrE can interact with AcrD, which does not possess its own PAP, and establish that the residues previously identified in AcrB binding are also involved in AcrD binding.

Methods: The acrD and acrE genes were expressed in a strain lacking acrABDEF (Δ3RND). PAP residues involved in promiscuous interactions were predicted based on previously defined PAP-RND interactions and corresponding mutations generated in acrA and acrE. Antimicrobial susceptibility of the mutant strains was determined.

Results: Co-expression of acrD and acrE significantly decreased susceptibility of the Δ3RND strain to AcrD substrates, showing that AcrE can form a functional complex with AcrD. The substrate profile of Salmonella AcrD differed from that of Escherichia coli AcrD. Mutations targeting the previously defined PAP-RND interaction sites in AcrA/AcrE impaired efflux of AcrD-dependent substrates.

Conclusions: These data indicate that AcrE forms an efflux-competent pump with AcrD and thus presents an alternative PAP for this pump. Mutagenesis of the conserved RND binding sites validates the interchangeability of AcrA and AcrE, highlighting them as potential drug targets for efflux inhibition.
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http://dx.doi.org/10.1093/jac/dkab237DOI Listing
July 2021

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria.

Chem Rev 2021 May 28;121(9):5479-5596. Epub 2021 Apr 28.

School of Life Sciences, University of Essex, Colchester, CO4 3SQ United Kingdom.

Tripartite efflux pumps and the related type 1 secretion systems (T1SSs) in Gram-negative organisms are diverse in function, energization, and structural organization. They form continuous conduits spanning both the inner and the outer membrane and are composed of three principal components-the energized inner membrane transporters (belonging to ABC, RND, and MFS families), the outer membrane factor channel-like proteins, and linking the two, the periplasmic adaptor proteins (PAPs), also known as the membrane fusion proteins (MFPs). In this review we summarize the recent advances in understanding of structural biology, function, and regulation of these systems, highlighting the previously undescribed role of PAPs in providing a common architectural scaffold across diverse families of transporters. Despite being built from a limited number of basic structural domains, these complexes present a staggering variety of architectures. While key insights have been derived from the RND transporter systems, a closer inspection of the operation and structural organization of different tripartite systems reveals unexpected analogies between them, including those formed around MFS- and ATP-driven transporters, suggesting that they operate around basic common principles. Based on that we are proposing a new integrated model of PAP-mediated communication within the conformational cycling of tripartite systems, which could be expanded to other types of assemblies.
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http://dx.doi.org/10.1021/acs.chemrev.1c00055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8277102PMC
May 2021

Quantitative real-time analysis of the efflux by the MacAB-TolC tripartite efflux pump clarifies the role of ATP hydrolysis within mechanotransmission mechanism.

Commun Biol 2021 04 22;4(1):493. Epub 2021 Apr 22.

Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, CNRS UMR 7099, Université de Paris, Paris, France.

Tripartite efflux pumps built around ATP-binding cassette (ABC) transporters are membrane protein machineries that perform vectorial export of a large variety of drugs and virulence factors from Gram negative bacteria, using ATP-hydrolysis as energy source. Determining the number of ATP molecules consumed per transport cycle is essential to understanding the efficiency of substrate transport. Using a reconstituted pump in a membrane mimic environment, we show that MacAB-TolC from Escherichia coli couples substrate transport to ATP-hydrolysis with high efficiency. Contrary to the predictions of the currently prevailing "molecular bellows" model of MacB-operation, which assigns the power stroke to the ATP-binding by the nucleotide binding domains of the transporter, by utilizing a novel assay, we report clear synchronization of the substrate transfer with ATP-hydrolysis, suggesting that at least some of the power stroke for the substrate efflux is provided by ATP-hydrolysis. Our findings narrow down the window for energy consumption step that results in substrate transition into the TolC-channel, expanding the current understanding of the efflux cycle of the MacB-based tripartite assemblies. Based on that we propose a modified model of the MacB cycle within the context of tripartite complex assembly.
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http://dx.doi.org/10.1038/s42003-021-01997-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062640PMC
April 2021

Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation.

Elife 2020 12 14;9. Epub 2020 Dec 14.

Institute of Microbiology and Infection, University of Birmingham, Edgbaston, United Kingdom.

The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.
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http://dx.doi.org/10.7554/eLife.62614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806268PMC
December 2020

Mutations in the TolC Periplasmic Domain Affect Substrate Specificity of the AcrAB-TolC Pump.

Front Mol Biosci 2020 21;7:166. Epub 2020 Jul 21.

School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

TolC and the other members of the outer membrane factor (OMF) family are outer membrane proteins forming trimeric channels that serve as a conduit for most actively effluxed substrates in Gram-negative bacteria by providing a key component in a multitude of tripartite efflux-pumps. Current models of tripartite pump assembly ascribe substrate selection to the inner-membrane transporter and periplasmic-adapter protein (PAP) assembly, suggesting that TolC is a passive, non-selective channel. While the membrane-embedded portion of the protein adopts a porin-like fold, the periplasmic domain of TolC presents a unique "alpha-barrel" architecture. This alpha-barrel consists of pseudo-continuous α-helices forming curved coiled-coils, whose tips form α-helical hairpins, relaxation of which results in a transition of TolC from a closed to an open-aperture state allowing effective efflux of substrates through its channel. Here, we analyzed the effects of site-directed mutations targeting the alpha-barrel of TolC, of the principal tripartite efflux-pump AcrAB-TolC, on the activity and specificity of efflux. Live-cell functional assays with these TolC mutants revealed that positions both at the periplasmic tip of, and partway up the TolC coiled-coil alpha-barrel domain are involved in determining the functionality of the complex. We report that mutations affecting the electrostatic properties of the channel, particularly the D371V mutation, significantly impact growth even in the absence of antibiotics, causing hyper-susceptibility to all tested efflux-substrates. These results suggest that inhibition of TolC functionality is less well-tolerated than deletion of , and such inhibition may have an antibacterial effect. Significantly and unexpectedly, we identified antibiotic-specific phenotypes associated with novel TolC mutations, suggesting that substrate specificity may not be determined solely by the transporter protein or the PAP, but may reside at least partially with the TolC-channel. Furthermore, some of the effects of mutations are difficult to reconcile with the currently prevalent tip-to-tip model of PAP-TolC interaction due to their location higher-up on the TolC alpha-barrel relative to the proposed PAP-docking sites. Taken together our results suggest a possible new role for TolC in vetting of efflux substrates, alongside its established role in tripartite complex assembly.
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http://dx.doi.org/10.3389/fmolb.2020.00166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396618PMC
July 2020

Cryo-EM Structure and Molecular Dynamics Analysis of the Fluoroquinolone Resistant Mutant of the AcrB Transporter from .

Microorganisms 2020 Jun 23;8(6). Epub 2020 Jun 23.

School of Life Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.

is an important genus of Gram-negative pathogens, treatment of which has become problematic due to increases in antimicrobial resistance. This is partly attributable to the overexpression of tripartite efflux pumps, particularly the constitutively expressed AcrAB-TolC. Despite its clinical importance, the structure of the AcrB transporter remained unknown to-date, with much of our structural understanding coming from the orthologue. Here, by taking advantage of the styrene maleic acid (SMA) technology to isolate membrane proteins with closely associated lipids, we report the very first experimental structure of AcrB transporter. Furthermore, this novel structure provides additional insight into mechanisms of drug efflux as it bears the mutation (G288D), originating from a clinical isolate of Typhimurium presenting an increased resistance to fluoroquinolones. Experimental data are complemented by state-of-the-art molecular dynamics (MD) simulations on both the wild type and G288D variant of AcrB. Together, these reveal several important differences with respect to the protein, providing insights into the role of the G288D mutation in increasing drug efflux and extending our understanding of the mechanisms underlying antibiotic resistance.
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http://dx.doi.org/10.3390/microorganisms8060943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355581PMC
June 2020

Outer membrane protein size and LPS O-antigen define protective antibody targeting to the Salmonella surface.

Nat Commun 2020 02 12;11(1):851. Epub 2020 Feb 12.

Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, B15 2TT, UK.

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.
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http://dx.doi.org/10.1038/s41467-020-14655-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015928PMC
February 2020

Identification of binding residues between periplasmic adapter protein (PAP) and RND efflux pumps explains PAP-pump promiscuity and roles in antimicrobial resistance.

PLoS Pathog 2019 12 26;15(12):e1008101. Epub 2019 Dec 26.

Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

Active efflux due to tripartite RND efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. These pumps are also essential for Gram-negative pathogens to cause infection and form biofilms. They consist of an inner membrane RND transporter; a periplasmic adaptor protein (PAP), and an outer membrane channel. The role of PAPs in assembly, and the identities of specific residues involved in PAP-RND binding, remain poorly understood. Using recent high-resolution structures, four 3D sites involved in PAP-RND binding within each PAP protomer were defined that correspond to nine discrete linear binding sequences or "binding boxes" within the PAP sequence. In the important human pathogen Salmonella enterica, these binding boxes are conserved within phylogenetically-related PAPs, such as AcrA and AcrE, while differing considerably between divergent PAPs such as MdsA and MdtA, despite overall conservation of the PAP structure. By analysing these binding sequences we created a predictive model of PAP-RND interaction, which suggested the determinants that may allow promiscuity between certain PAPs, but discrimination of others. We corroborated these predictions using direct phenotypic data, confirming that only AcrA and AcrE, but not MdtA or MsdA, can function with the major RND pump AcrB. Furthermore, we provide functional validation of the involvement of the binding boxes by disruptive site-directed mutagenesis. These results directly link sequence conservation within identified PAP binding sites with functional data providing mechanistic explanation for assembly of clinically relevant RND-pumps and explain how Salmonella and other pathogens maintain a degree of redundancy in efflux mediated resistance. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a predictive model of pump-contacts that could be exploited in the future for the development of targeted therapeutics and efflux pump inhibitors.
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http://dx.doi.org/10.1371/journal.ppat.1008101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6975555PMC
December 2019

The mycolic acid reductase Rv2509 has distinct structural motifs and is essential for growth in slow-growing mycobacteria.

Mol Microbiol 2020 02 17;113(2):521-533. Epub 2019 Dec 17.

School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK.

The final step in mycolic acid biosynthesis in Mycobacterium tuberculosis is catalysed by mycolyl reductase encoded by the Rv2509 gene. Sequence analysis and homology modelling indicate that Rv2509 belongs to the short-chain fatty acid dehydrogenase/reductase (SDR) family, but with some distinct features that warrant its classification as belonging to a novel family of short-chain dehydrogenases. In particular, the predicted structure revealed a unique α-helical C-terminal region which we demonstrated to be essential for Rv2509 function, though this region did not seem to play any role in protein stabilisation or oligomerisation. We also show that unlike the M. smegmatis homologue which was not essential for growth, Rv2509 was an essential gene in slow-growing mycobacteria. A knockdown strain of the BCG2529 gene, the Rv2509 homologue in Mycobacterium bovis BCG, was unable to grow following the conditional depletion of BCG2529. This conditional depletion also led to a reduction of mature mycolic acid production and accumulation of intermediates derived from 3-oxo-mycolate precursors. Our studies demonstrate novel features of the mycolyl reductase Rv2509 and outline its role in mycobacterial growth, highlighting its potential as a new target for therapies.
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http://dx.doi.org/10.1111/mmi.14437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065075PMC
February 2020

Editorial: Bacterial Mechanisms of Antibiotic Resistance: A Structural Perspective.

Front Mol Biosci 2019 14;6:71. Epub 2019 Aug 14.

Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, United States.

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http://dx.doi.org/10.3389/fmolb.2019.00071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702466PMC
August 2019

Structure-Guided Identification of a Nonhuman Morbillivirus with Zoonotic Potential.

J Virol 2018 12 12;92(23). Epub 2018 Nov 12.

The Pirbright Institute, Surrey, United Kingdom

Morbilliviruses infect a broad range of mammalian hosts, including ruminants, carnivores, and humans. The recent eradication of rinderpest virus (RPV) and the active campaigns for eradication of the human-specific measles virus (MeV) have raised significant concerns that the remaining morbilliviruses may emerge in so-called vacated ecological niches. Seeking to assess the zoonotic potential of nonhuman morbilliviruses within human populations, we found that peste des petits ruminants virus (PPRV)-the small-ruminant morbillivirus-is restricted at the point of entry into human cells due to deficient interactions with human SLAMF1-the immune cell receptor for morbilliviruses. Using a structure-guided approach, we characterized a single amino acid change, mapping to the receptor-binding domain in the PPRV hemagglutinin (H) protein, which overcomes this restriction. The same mutation allowed escape from some cross-protective, human patient, anti-MeV antibodies, raising concerns that PPRV is a pathogen with zoonotic potential. Analysis of natural variation within human and ovine SLAMF1 also identified polymorphisms that could correlate with disease resistance. Finally, the mechanistic nature of the PPRV restriction was also investigated, identifying charge incompatibility and steric hindrance between PPRV H and human SLAMF1 proteins. Importantly, this research was performed entirely using surrogate virus entry assays, negating the requirement for derivation of a human-tropic PPRV and illustrating alternative strategies for identifying gain-of-function mutations in viral pathogens. A significant proportion of viral pandemics occur following zoonotic transmission events, where animal-associated viruses jump species into human populations. In order to provide forewarnings of the emergence of these viruses, it is necessary to develop a better understanding of what determines virus host range, often at the genetic and structural levels. In this study, we demonstrated that the small-ruminant morbillivirus, a close relative of measles, is unable to use human receptors to enter cells; however, a change of a single amino acid in the virus is sufficient to overcome this restriction. This information will be important for monitoring this virus's evolution in the field. Of note, this study was undertaken , without generation of a fully infectious virus with this phenotype.
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http://dx.doi.org/10.1128/JVI.01248-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232486PMC
December 2018

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.

Int J Mol Sci 2018 Mar 27;19(4). Epub 2018 Mar 27.

Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan.

ATP-binding cassette transporter VcaM ( ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.
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http://dx.doi.org/10.3390/ijms19041000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979437PMC
March 2018

A cytosolic copper storage protein provides a second level of copper tolerance in Streptomyces lividans.

Metallomics 2018 01;10(1):180-193

School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK.

Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P-type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.
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http://dx.doi.org/10.1039/c7mt00299hDOI Listing
January 2018

Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS.

J Bacteriol 2017 09 22;199(18). Epub 2017 Aug 22.

School of Biosciences, University of Birmingham, Birmingham, United Kingdom

The EvgS/EvgA two-component system of is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this. One of the ways bacteria sense their environment is through two-component systems, which have one membrane-bound protein to do the sensing and another inside the cell to turn genes on or off in response to what the membrane-bound protein has detected. The membrane-bound protein must thus be able to detect the stress and signal this detection event to the protein inside the cell. To understand this process, we studied a protein that helps to survive exposure to low pH, which it must do before taking up residence in the gastrointestinal tract. We describe a predicted structure for the main sensing part of the protein and identify some key residues within it that are involved in the sensing and signaling processes. We propose a mechanism for how the protein may become activated and present some evidence to support our proposal.
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http://dx.doi.org/10.1128/JB.00310-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573083PMC
September 2017

Oxidative footprinting in the study of structure and function of membrane proteins: current state and perspectives.

Biochem Soc Trans 2015 Oct;43(5):983-94

Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, U.S.A.

Membrane proteins, such as receptors, transporters and ion channels, control the vast majority of cellular signalling and metabolite exchange processes and thus are becoming key pharmacological targets. Obtaining structural information by usage of traditional structural biology techniques is limited by the requirements for the protein samples to be highly pure and stable when handled in high concentrations and in non-native buffer systems, which is often difficult to achieve for membrane targets. Hence, there is a growing requirement for the use of hybrid, integrative approaches to study the dynamic and functional aspects of membrane proteins in physiologically relevant conditions. In recent years, significant progress has been made in the field of oxidative labelling techniques and in particular the X-ray radiolytic footprinting in combination with mass spectrometry (MS) (XF-MS), which provide residue-specific information on the solvent accessibility of proteins. In combination with both low- and high-resolution data from other structural biology approaches, it is capable of providing valuable insights into dynamics of membrane proteins, which have been difficult to obtain by other structural techniques, proving a highly complementary technique to address structure and function of membrane targets. XF-MS has demonstrated a unique capability for identification of structural waters and conformational changes in proteins at both a high degree of spatial and a high degree of temporal resolution. Here, we provide a perspective on the place of XF-MS among other structural biology methods and showcase some of the latest developments in its usage for studying water-mediated transmembrane (TM) signalling, ion transport and ligand-induced allosteric conformational changes in membrane proteins.
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http://dx.doi.org/10.1042/BST20150130DOI Listing
October 2015

Modular Design of the Selectivity Filter Pore Loop in a Novel Family of Prokaryotic 'Inward Rectifier' (NirBac) channels.

Sci Rep 2015 Oct 16;5:15305. Epub 2015 Oct 16.

Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, United Kingdom.

Potassium channels exhibit a modular design with distinct structural and functional domains; in particular, a highly conserved pore-loop sequence that determines their ionic selectivity. We now report the functional characterisation of a novel group of functionally non-selective members of the prokaryotic 'inward rectifier' subfamily of K(+) channels. These channels share all the key structural domains of eukaryotic and prokaryotic Kir/KirBac channels, but instead possess unique pore-loop selectivity filter sequences unrelated to any other known ionic selectivity filter. The strikingly unusual architecture of these 'NirBac' channels defines a new family of functionally non-selective ion channels, and also provides important insights into the modular design of ion channels, as well as the evolution of ionic selectivity within this superfamily of tetrameric cation channels.
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http://dx.doi.org/10.1038/srep15305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607889PMC
October 2015

Architecture and roles of periplasmic adaptor proteins in tripartite efflux assemblies.

Front Microbiol 2015 28;6:513. Epub 2015 May 28.

Institute of Microbiology and Infection, University of Birmingham Birmingham, UK.

Recent years have seen major advances in the structural understanding of the different components of tripartite efflux assemblies, which encompass the multidrug efflux (MDR) pumps and type I secretion systems. The majority of these investigations have focused on the role played by the inner membrane transporters and the outer membrane factor (OMF), leaving the third component of the system - the Periplasmic Adaptor Proteins (PAPs) - relatively understudied. Here we review the current state of knowledge of these versatile proteins which, far from being passive linkers between the OMF and the transporter, emerge as active architects of tripartite assemblies, and play diverse roles in the transport process. Recognition between the PAPs and OMFs is essential for pump assembly and function, and targeting this interaction may provide a novel avenue for combating multidrug resistance. With the recent advances elucidating the drug efflux and energetics of the tripartite assemblies, the understanding of the interaction between the OMFs and PAPs is the last piece remaining in the complete structure of the tripartite pump assembly puzzle.
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http://dx.doi.org/10.3389/fmicb.2015.00513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446572PMC
June 2015

Cross-species chimeras reveal BamA POTRA and β-barrel domains must be fine-tuned for efficient OMP insertion.

Mol Microbiol 2015 Aug 6;97(4):646-59. Epub 2015 Jun 6.

Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains at its N-terminus. The C-terminus of BamA folds into a β-barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram-negative bacteria and appear to function in a species-specific manner. Here we investigate the nature of this species-specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the β-barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the β-barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the β-barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.
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http://dx.doi.org/10.1111/mmi.13052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4950039PMC
August 2015

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

Proc Natl Acad Sci U S A 2015 Mar 3;112(11):3511-6. Epub 2015 Mar 3.

Antimicrobials Research Group, School of Immunity and Infection, College of Medical and Dental Sciences, Institute of Microbiology and Infection, The University of Birmingham, Birmingham B15 2TT, United Kingdom;

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.
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http://dx.doi.org/10.1073/pnas.1419939112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371985PMC
March 2015

Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa.

Biochimie 2015 Apr 7;111:58-69. Epub 2015 Feb 7.

Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense 400, São Carlos, SP 13560-970, Brazil. Electronic address:

Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops excelsa (CEP), which has a high pH and thermal stability, is no exception. To date, the structural and molecular events underscoring such biochemical behavior have not been explored in depth. In order to identify the structural characteristics accounting for the high stability of palm tree peroxidases, we solved and refined the X-ray structure of native CEP at a resolution of 2.6 Å. The CEP structure has an overall fold typical of plant peroxidases and confirmed the conservation of characteristic structural elements such as the heme group and calcium ions. At the same time the structure revealed important modifications in the amino acid residues in the vicinity of the exposed heme edge region, involved in substrate binding, that could account for the morphological variations among palm tree peroxidases through the disruption of molecular interactions at the second binding site. These modifications could alleviate the inhibition of enzymatic activity caused by molecular interactions at the latter binding site. Comparing the CEP crystallographic model described here with other publicly available peroxidase structures allowed the identification of a noncovalent homodimer assembly held together by a number of ionic and hydrophobic interactions. We demonstrate, that this dimeric arrangement results in a more stable protein quaternary structure through stabilization of the regions that are highly dynamic in other peroxidases. In addition, we resolved five N-glycosylation sites, which might also contribute to enzyme stability and resistance against proteolytic cleavage.
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http://dx.doi.org/10.1016/j.biochi.2015.01.014DOI Listing
April 2015

Genetic assessment of the role of AcrB β-hairpins in the assembly of the TolC-AcrAB multidrug efflux pump of Escherichia coli.

Mol Microbiol 2014 Mar 21;91(5):965-75. Epub 2014 Jan 21.

School of Life Sciences, Arizona State University, Tempe, AZ, 85287, USA; Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, OK, 73019, USA.

The tripartite AcrAB-TolC multidrug efflux pump of Escherichia coli is the central conduit for cell-toxic compounds and contributes to antibiotic resistance. While high-resolution structures of all three proteins have been solved, much remains to be learned as to how the individual components come together to form a functional complex. In this study, we investigated the importance of the AcrB β-hairpins belonging to the DN and DC subdomains, which are presumed to dock with TolC, in complex stability and activity of the complete pump. Our data show that the DN subdomain β-hairpin residues play a more critical role in complex stability and activity than the DC subdomain hairpin residues. The failure of the AcrB DN β-hairpin deletion mutant to engage with TolC leads to the drug hypersensitivity phenotype, which is reversed by compensatory alterations in the lipoyl and β-barrel domains of AcrA. Moreover, AcrA and TolC mutants that induce TolC opening also reverse the drug hypersensitivity phenotype of the AcrB β-hairpin mutants, indicating a failure by the AcrB mutant to interact and thus induce TolC opening on its own. Together, these data suggest that both AcrB β-hairpins and AcrA act to stabilize the tripartite complex and induce TolC opening for drug expulsion.
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http://dx.doi.org/10.1111/mmi.12508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3984920PMC
March 2014

Mutational and topological analysis of the Escherichia coli BamA protein.

PLoS One 2013 23;8(12):e84512. Epub 2013 Dec 23.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

The multi-protein β-barrel assembly machine (BAM) of Escherichia coli is responsible for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains in its N-terminus. The C-terminus of BamA contains a β-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA β-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify β-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA β-barrel are essential.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084512PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3871556PMC
October 2014

Control of KirBac3.1 potassium channel gating at the interface between cytoplasmic domains.

J Biol Chem 2014 Jan 20;289(1):143-51. Epub 2013 Nov 20.

From the Biological Physics Group, Clarendon Laboratory, University of Oxford, Oxford OX1 3PU, United Kingdom.

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating.
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http://dx.doi.org/10.1074/jbc.M113.501833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879539PMC
January 2014

Tripartite efflux pumps: energy is required for dissociation, but not assembly or opening of the outer membrane channel of the pump.

Mol Microbiol 2013 May 9;88(3):590-602. Epub 2013 Apr 9.

School of Biological and Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, UK.

The MtrCDE multidrug pump, from Neisseria gonorrhoeae, is assembled from the inner and outer membrane proteins MtrD and MtrE, which are connected by the periplasmic membrane fusion protein MtrC. Although it is clear that MtrD delivers drugs to the channel of MtrE, it remains unclear how drug delivery and channel opening are connected. We used a vancomycin sensitivity assay to test for opening of the MtrE channel. Cells expressing MtrE or MtrE-E434K were insensitive to vancomycin; but became moderately and highly sensitive to vancomycin respectively, when coexpressed with MtrC, suggesting that the MtrE channel opening requires MtrC binding and is energy-independent. Cells expressing wild-type MtrD, in an MtrCE background, were vancomycin-insensitive, but moderately sensitive in an MtrCE-E434K background. The mutation of residues involved in proton translocation inactivated MtrD and abolished drug efflux, rendered both MtrE and MtrE-E434K vancomycin-insensitive; imply that the pump-component interactions are preserved, and that the complex is stable in the absence of proton flux, thus sealing the open end of MtrE. Following the energy-dependent dissociation of the tripartite complex, the MtrE channel is able to reseal, while MtrE-E434K is unable to do so, resulting in the vancomycin-sensitive phenotype. Thus, our findings suggest that opening of the OMP via interaction with the MFP is energy-independent, while both drug export and complex dissociation require active proton flux.
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http://dx.doi.org/10.1111/mmi.12211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664412PMC
May 2013

Structure of a KirBac potassium channel with an open bundle crossing indicates a mechanism of channel gating.

Nat Struct Mol Biol 2012 Jan 8;19(2):158-63. Epub 2012 Jan 8.

Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK.

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying (Kir) potassium channels, and recent crystal structures of both Kir and KirBac channels have provided major insight into their unique structural architecture. However, all of the available structures are closed at the helix bundle crossing, and therefore the structural mechanisms that control opening of their primary activation gate remain unknown. In this study, we engineered the inner pore-lining helix (TM2) of KirBac3.1 to trap the bundle crossing in an apparently open conformation and determined the crystal structure of this mutant channel to 3.05 Å resolution. Contrary to previous speculation, this new structure suggests a mechanistic model in which rotational 'twist' of the cytoplasmic domain is coupled to opening of the bundle-crossing gate through a network of inter- and intrasubunit interactions that involve the TM2 C-linker, slide helix, G-loop and the CD loop.
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http://dx.doi.org/10.1038/nsmb.2208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272479PMC
January 2012

Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.

J Biol Chem 2011 Jul 24;286(30):26900-12. Epub 2011 May 24.

School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, United Kingdom.

The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE(3)MtrC(6)), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.
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http://dx.doi.org/10.1074/jbc.M111.246595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143649PMC
July 2011

Opening of the outer membrane protein channel in tripartite efflux pumps is induced by interaction with the membrane fusion partner.

J Biol Chem 2011 Feb 29;286(7):5484-93. Epub 2010 Nov 29.

School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, United Kingdom.

The multiple transferable resistance (MTR) pump, from Neisseria gonorrhoeae, is typical of the specialized machinery used to translocate drugs across the inner and outer membranes of Gram-negative bacteria. It consists of a tripartite complex composed of an inner-membrane transporter, MtrD, a periplasmic membrane fusion protein, MtrC, and an outer-membrane channel, MtrE. We have expressed the components of the pump in Escherichia coli and used the antibiotic vancomycin, which is too large to cross the outer-membrane by passive diffusion, to test for opening of the MtrE channel. Cells expressing MtrCDE are not susceptible to vancomycin, indicating that the channel is closed; but become susceptible to vancomycin in the presence of transported substrates, consistent with drug-induced opening of the MtrE channel. A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin. Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin. However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.
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http://dx.doi.org/10.1074/jbc.M110.187658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037662PMC
February 2011

Ion mobility mass spectrometry of two tetrameric membrane protein complexes reveals compact structures and differences in stability and packing.

J Am Chem Soc 2010 Nov;132(44):15468-70

Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK.

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.
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http://dx.doi.org/10.1021/ja104312eDOI Listing
November 2010

Functional complementation and genetic deletion studies of KirBac channels: activatory mutations highlight gating-sensitive domains.

J Biol Chem 2010 Dec 28;285(52):40754-61. Epub 2010 Sep 28.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PU, United Kingdom.

The superfamily of prokaryotic inwardly rectifying (KirBac) potassium channels is homologous to mammalian Kir channels. However, relatively little is known about their regulation or about their physiological role in vivo. In this study, we have used random mutagenesis and genetic complementation in K(+)-auxotrophic Escherichia coli and Saccharomyces cerevisiae to identify activatory mutations in a range of different KirBac channels. We also show that the KirBac6.1 gene (slr5078) is necessary for normal growth of the cyanobacterium Synechocystis PCC6803. Functional analysis and molecular dynamics simulations of selected activatory mutations identified regions within the slide helix, transmembrane helices, and C terminus that function as important regulators of KirBac channel activity, as well as a region close to the selectivity filter of KirBac3.1 that may have an effect on gating. In particular, the mutations identified in TM2 favor a model of KirBac channel gating in which opening of the pore at the helix-bundle crossing plays a far more important role than has recently been proposed.
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http://dx.doi.org/10.1074/jbc.M110.175687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003375PMC
December 2010

Conformational changes during the gating of a potassium channel revealed by structural mass spectrometry.

Structure 2010 Jul;18(7):839-46

Center for Synchrotron Biosciences, Case Western Reserve University, Cleveland, OH 44022, USA.

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.
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http://dx.doi.org/10.1016/j.str.2010.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124773PMC
July 2010
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