mass spectrometry (MS) is emerging as an invaluable
technique to probe the structure, interactions, and dynamics of membrane
proteins (MPs). However, maintaining native-like MP conformations
in the gas phase using detergent solubilized proteins is often challenging
and may limit structural analysis. Amphipols, such as the well characterized
A8-35, are alternative reagents able to maintain the solubility of
MPs in detergent-free solution. In this work, the ability of A8-35
to retain the structural integrity of MPs for interrogation by electrospray
ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)
is compared systematically with the commonly used detergent dodecylmaltoside.
MPs from the two major structural classes were selected for analysis,
including two β-barrel outer MPs, PagP and OmpT (20.2 and 33.5
kDa, respectively), and two α-helical proteins, Mhp1 and GalP
(54.6 and 51.7 kDa, respectively). Evaluation of the rotationally
averaged collision cross sections of the observed ions revealed that
the native structures of detergent solubilized MPs were not always
retained in the gas phase, with both collapsed and unfolded species
being detected. In contrast, ESI-IMS-MS analysis of the amphipol solubilized
MPs studied resulted in charge state distributions consistent with
less gas phase induced unfolding, and the presence of lowly charged
ions which exhibit collision cross sections comparable with those
calculated from high resolution structural data. The data demonstrate
that A8-35 can be more effective than dodecylmaltoside at maintaining
native MP structure and interactions in the gas phase, permitting
noncovalent ESI-IMS-MS analysis of MPs from the two major structural
classes, while gas phase dissociation from dodecylmaltoside micelles
leads to significant gas phase unfolding, especially for the α-helical
Phylogenetic classification divides the major facilitator superfamily (MFS) into 82 families, including 25 families that are comprised of transporters with no characterized functions. This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a member of the “unknown major facilitator family-2” (UMF-2). BC3310 was shown to be a multidrug eﬄux pump conferring resistance to ethidium bromide, SDS and silver nitrate when heterologously expressed in Escherichia coli DH5α ΔacrAB. A conserved aspartate residue (D105) in putative transmembrane helix 4 was identified, which was essential for the energy dependent ethidium bromide eﬄux by BC3310. Transport proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF-2 proteins revealed that they carry a variant of the MFS motif A, which may be used as a marker to distinguish easily between this family and other MFS proteins. Genes orthologous to bc3310 are highly conserved within the B. cereus group of organisms and thus belong to the core genome, suggesting an important conserved functional role in the normal physiology of these bacteria.
MFS; drug resistance; eﬄux protein; Bacillus cereus; UMF-2
The era of antibiotics as a cure-all for bacterial infections appears to be coming to an end. The emergence of multidrug resistance in many hospital-associated pathogens has resulted in “superbugs” that are effectively untreatable. Multidrug eﬄux pumps are well known mediators of bacterial drug resistance. Genome sequencing efforts have highlighted an abundance of putative eﬄux pump genes in bacteria. However, it is not clear how many of these pumps play a role in antimicrobial resistance. Eﬄux pump genes that participate in drug resistance can be under tight regulatory control and expressed only in response to substrates. Consequently, changes in gene expression following antimicrobial shock may be used to identify eﬄux pumps that mediate antimicrobial resistance. Using this approach we have characterized several novel eﬄux pumps in bacteria. In one example we recently identified the Acinetobacter
chlorhexidine eﬄux protein (AceI) eﬄux pump in Acinetobacter. AceI is a prototype for a novel family of multidrug eﬄux pumps conserved in many proteobacterial lineages. The discovery of this family raises the possibility that additional undiscovered intrinsic resistance proteins may be encoded in the core genomes of pathogenic bacteria.
multidrug eﬄux systems; bacterial transmembrane pair; adaptive resistance; bacterial drug resistance transcriptomics
Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins.
Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug efflux systems which are very common in Proteobacteria: the proteobacterial antimicrobial compound efflux (PACE) family. This is the first new family of multidrug efflux pumps to be described in 15 years.
The gene hyuP from Microbacterium liquefaciens AJ 3912 with an added His6 tag was cloned into the expression plasmid pTTQ18 in an Escherichia coli host strain. The transformed E. coli showed transport of radioisotope-labeled 5-substituted hydantoins with apparent Km values in the micromolar range. This activity exhibited a pH optimum of 6.6 and was inhibited by dinitrophenol, indicating the requirement of energy for the transport system. 5-Indolyl methyl hydantoin and 5-benzyl hydantoin were the preferred substrates, with selectivity for a hydrophobic substituent in position 5 of hydantoin and for the l isomer over the d isomer. Hydantoins with less hydrophobic substituents, cytosine, thiamine, uracil, allantoin, adenine, and guanine, were not effective ligands. The His-tagged hydantoin transport protein was located in the inner membrane fraction, from which it was solubilized and purified and its identity was authenticated.
We have developed a simple native-like surface-tethered membrane system to investigate the activity of cytochrome bo3 (cbo3), a terminal oxidase in Escherichia coli. The tethered membranes consist of E. coli inner membrane extracts mixed with additional E. coli lipids containing various amounts of the cbo3 substrate ubiquinol-10 (UQ-10). Tethered membranes are formed by self assembly from vesicles onto gold electrodes functionalised with cholesterol derivatives. Cytochrome bo3 activity was monitored using cyclic voltammetry with electron transfer to cbo3 mediated by UQ-10. The apparent KM for oxygen with this system is 1.1±0.4 μM, in good agreement with literature values for whole cell experiments and for purified cbo3. Increasing the concentration of lipophilic UQ-10 in the membrane leads to an increase in cbo3 activity. The activity of cbo3 with long chain ubiquinones appears to be different to previous reports using short chain substrate analogues such as UQ-1 in that typical Michaelis Menten kinetics are not observed using UQ-10. This native-like membrane model thus provides new insights into the interaction of transmembrane enzymes with hydrophobic substrates which contrasts with studies using hydrophilic UQ analogues.
ubiquinol oxidase; tethered bilayer; cytochrome bo3; quinone pool
Mhp1, a hydantoin transporter from M. liquefaciens, was purified and crystallized. Diffraction data were collected to 2.85 Å resolution; the crystal belonged to the orthorhombic space group P212121.
The integral membrane protein Mhp1 from Microbacterium liquefaciens transports hydantoins and belongs to the nucleobase:cation symporter 1 family. Mhp1 was successfully purified and crystallized. Initial crystals were obtained using the hanging-drop vapour-diffusion method but diffracted poorly. Optimization of the crystallization conditions resulted in the generation of orthorhombic crystals (space group P212121, unit-cell parameters a = 79.7, b = 101.1, c = 113.8 Å). A complete data set has been collected from a single crystal to a resolution of 2.85 Å with 64 741 independent observations (94% complete) and an R
merge of 0.12. Further experimental phasing methods are under way.
transporters; nucleobase:cation symporter 1 family; membrane proteins; hydantoins
Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential.
Fifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H+-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials.
Here, we describe an efficient strategy for heterologous production of membrane transport proteins in E. coli. Small-volume cultures (10 mL) produced sufficient amount of proteins to assess their purity and aggregation state. The methods described in this work are simple to implement and can be easily applied to many more membrane proteins.
The inner membrane of Escherichia coli, over-expressing an ubiquinol oxidase, cytochrome bo3 (cbo3), was tethered in a planar configuration to a gold electrode. Electron transfer to cbo3 was achieved via native ubiquinol-8 or added ubiquinol-10 and impedance spectroscopy was used to characterise the diffusion properties of the ubiquinol/ubiquinone in the tethered membrane system. Spectra were obtained at varying DC potentials covering the potential window in which the voltammetric catalytic wave of cbo3 is visible. These spectra were compared to those obtained after addition of a potent inhibitor of cbo3, cyanide, and the difference in impedance was analysed using a derived equivalent circuit, which is similar to that of Open Finite-Length Diffusion (OFLD) or the finite Warburg circuit, but with the boundary conditions modified to account for the fact that ubiquinol reoxidation is limited by enzyme activity. Analysis of the impedance spectra of the tethered membrane system gave kinetic parameters that are consistent with values obtained using cyclic voltammetry. Importantly, the diffusion rate of ubiquinone (10−13 - 10−12 cm2/s) was found to be orders of magnitude lower than accepted values for lateral diffusion (10−8 - 10−7 cm2/s). It is hypothesised that this result represent perpendicular diffusion of quinone across the membrane, corresponding to a ‘flip’ time between 0.05 and 1 s.
A novel electrochemical approach is described for redox-active membrane proteins. A total membrane extract (in the form of vesicles) of Bacillus subtilis is tethered onto gold surfaces modified with cholesterol based thiols. The membrane vesicles remain intact on the surface and do not rupture or fuse to form a planar bilayer. Oxidation/reduction signals are obtained of the natural co-enzyme, menaquinone-7, located in the membrane. The membrane protein, succinate menaquinone oxidoreductase (SQR), remains in the vesicles and is able to reduce fumarate using menaquinone as mediator. The catalysis of the reverse reaction (oxidation of succinate), which is the natural catalytic function of SQR, is almost absent with menaquinone. However, adding the co-enzyme ubiquinone, which has a reduction potential that is about 0.2 V higher, restores the succinate oxidation activity.
An electrode surface is presented that enables the characterisation of redox-active membrane enzymes in a native-like environment. An ubiquinol oxidase from Escherichia coli, cytochrome bo3 (cbo3), has been co-immobilised into tethered bilayer lipid membranes (tBLMs). The tBLM is formed on gold surfaces functionalised with cholesterol tethers which insert into the lower leaflet of the membrane. The planar membrane architecture is formed by self assembly of proteoliposomes and its structure is characterised by surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS) and tapping-mode atomic force microscopy (TM-AFM). The functionality of cbo3 is investigated by cyclic voltammetry (CV) and is confirmed by the catalytic reduction of oxygen. Interfacial electron transfer to cbo3 is mediated by the membrane-localised ubiquinol-8, the physiological electron donor of cbo3. Enzyme coverages observed with TM-AFM and CV coincide (2–8.5 fmol·cm−2) indicating that most - if not all - cbo3 on the surface is catalytically active and thus retains its integrity during immobilisation.
Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport (Cloherty, E. K., Levine, K. B., & Carruthers, A. (2001). The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry, 40(51), 15549–15561). This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, endofacial binding sites for CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogs, asks what structural features of endofacial site ligands determine binding site affinity and cooperativity. Like CB, FSK competitively inhibits exchange 3-O-methylglucose transport (sugar uptake in cells containing intracellular sugar) but non-competitively inhibits sugar uptake into cells lacking sugar at 4°C. This refutes the hypothesis that FSK binds at GLUT1 endofacial and exofacial sugar binding sites. Some forskolin derivatives and cytochalasins inhibit equilibrium [3H]-CB binding to red cell membranes depleted of peripheral proteins at 4°C. Others produce a moderate stimulation of [3H]-CB binding when introduced at low concentrations but inhibit binding as their concentration is increased. Yet other analogs modestly stimulate [3H]-CB binding at all inhibitor concentrations applied. These findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of models for GLUT1-mediated sugar transport and GLUT1 quaternary structure and we evaluate the major determinants of ligand binding affinity and cooperativity.
Transcriptional profiling highlighted a subset of genes encoding putative multidrug transporters in the pathogen Bacillus cereus that were up-regulated during stress produced by bile salts. One of these multidrug transporters (BC4707) was selected for investigation. Functional characterization of the BC4707 protein in Escherichia coli revealed a role in the energized efflux of xenobiotics. Phenotypic analyses after inactivation of the gene bc4707 in Bacillus cereus ATCC14579 suggested a more specific, but modest role in the efflux of norfloxacin. In addition to this, transcriptional analyses showed that BC4707 is also expressed during growth of B. cereus under non-stressful conditions where it may have a role in the normal physiology of the bacteria. Altogether, the results indicate that bc4707, which is part of the core genome of the B. cereus group of bacteria, encodes a multidrug resistance efflux protein that is likely involved in maintaining intracellular homeostasis during growth of the bacteria.
Crystal structures of a membrane protein transporter in three different conformational states provide insights into the transport mechanism.
Secondary active transporters move molecules across cell membranes by coupling this process to the energetically favourable downhill movement of ions or protons along an electrochemical gradient. They function by the alternating access model of transport in which, through conformational changes, the substrate binding site alternately faces either side of the membrane. Owing to the difficulties in obtaining the crystal structure of a single transporter in different conformational states, relatively little structural information is known to explain how this process occurs. Here, the structure of the sodium-benzylhydantoin transporter, Mhp1, from Microbacterium liquefaciens, has been determined in three conformational states; from this a mechanism is proposed for switching from the outward-facing open conformation through an occluded structure to the inward-facing open state.
membrane transport; transport protein; alternating access; hydantoins
The structure of the sodium-benzylhydantoin transport protein, Mhp1, from Microbacterium liquefaciens comprises a 5-helix inverted repeat, which is widespread amongst secondary transporters. Here we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 Å resolution, complementing its previously-described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward- facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8 and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.
The ‘Nucleobase-Cation-Symport-1’, NCS1, transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85 Å resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, ten of which are arranged in two inverted repeats of 5 helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine (LeuTAa) and the galactose (vSGLT) transporters reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronised by the inverted repeat helices 3 and 8, providing the structural basis of the ‘alternating access’ model for membrane transport.
Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of β-glucuronides with synthetic [14C]phenyl-1-thio-β-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of β-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.