Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer

The small multidrug resistance family of transporters is widespread in bacteria and is responsible for bacterial resistance to toxic aromatic cations by proton-linked efflux. We have determined the three-dimensional (3D) structure of the Escherichia coli multidrug transporter EmrE by electron cryomicroscopy of 2D crystals, including data to 7.0 A resolution. The structure of EmrE consists of a bundle of eight transmembrane alpha-helices with one substrate molecule bound near the centre. The substrate binding chamber is formed from six helices and is accessible both from the aqueous phase and laterally from the lipid bilayer. The most remarkable feature of the structure of EmrE is that it is an asymmetric homodimer. The possible arrangement of the two polypeptides in the EmrE dimer is discussed based on the 3D density map.     

Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding

P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the three-dimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of approximately 2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.     

Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 x 106 x 162 A. It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR.anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain.     

SecB modulates the nucleotide-bound state of SecA and stimulates ATPase activity

In Escherichia coli, the formation of SecA-SecB complexes has a direct effect on SecA ATPase activity. The mechanism of this interaction was evaluated and defined using controlled trypsinolysis, equilibrium dialysis at low temperature, and kinetic analyses of the SecA ATPase reaction. The proteolysis data indicate that SecB and the nonhydrolyzable ATP analogue AMP-P-C-P induce similar conformational changes in SecA which result in a more open or extended structure that is suggestive of the ATP-bound form. The effect is synergistic and concentration-dependent, and requires the occupation of both the high- and low-affinity nucleotide binding sites for maximum effect. The equilibrium dialysis experiments and kinetic data support the observation that the SecB-enhanced SecA ATPase activity is the result of an increased rate of ATP hydrolysis rather than an increase in the affinity of ATP for SecA and that the high-affinity nucleotide binding site is conformationally regulated by SecB. It appears that SecB may function as an intermolecular regulator of ATP hydrolysis by promoting the ATP-bound state of SecA. The inhibition of SecA ATPase activity by sodium azide in the presence of IMVs and a functional signal peptide further indicates that SecB promotes the ATP-bound form of SecA.     

Molecular dynamics simulations of LysRS: an asymmetric state

We report molecular dynamics simulations of the Escherichia coli Lysyl-tRNA synthetase LysU isoform carried out as a benchmark for mutant simulations in in silico protein engineering efforts. Unlike previous studies of aminoacyl-tRNA synthetases, LysU is modelled in its full dimeric form with explicit solvent. While developing a suitable simulation protocol, we observed an asymmetry that persists despite improvements to the model. This prediction has directly led to experiments that establish a functional asymmetry in nucleotide binding by LysU. The development of a simulation protocol and validation of the model are presented here. The observed asymmetry is described and the role of protein flexibility in developing the asymmetry is discussed.     

New insights into the structure and oligomeric state of the bacterial multidrug transporter EmrE: an unusual asymmetric homo-dimer

EmrE is a small multidrug transporter that contains 110 amino acid residues that form four transmembrane alpha-helices. The three-dimensional structure of EmrE has been determined from two-dimensional crystals by electron cryo-microscopy. EmrE is an asymmetric homo-dimer with one substrate molecule bound in a chamber accessible laterally from one leaflet of the lipid bilayer. Evidence from substrate binding analyses and analytical ultracentrifugation of detergent-solubilised EmrE shows that the minimum functional unit for substrate binding is a dimer. However, it is possible that EmrE exists as a tetramer in vivo and plausible models are suggested based upon analyses of two-dimensional crystals.     

Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles

The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu(2-30+), a 36-residue polypeptide containing residues 2-30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional (1)H/(15)N PISEMA spectra of uniformly (15)N-labeled Vpu(2-30+) obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a (1)H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane alpha-helix spanning residues 8-25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent (15)N chemical shift and (1)H-(15)N dipolar coupling frequencies. Tilt angles of 30 degrees and 21 degrees are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers.     

Three-dimensional structure of d(GGGATCCC) in the crystalline state.

The structure of the self-complementary octamer d(GGGATCCC) has been analysed by single crystal X-ray diffraction methods at a nominal resolution of 2.5 A. With acceptable stereochemistry of the model the crystallographic R factor was 16.6% after restrained least-squares refinement. In the crystal, d(GGGATCCC) forms an A-DNA double helix with slightly varying conformation of the two strands. The average displacement of the base pairs from the helix axis is unusually large and is accompanied by pronounced sliding of the base pairs along their long axes at all dinucleotide steps except for the central AT. With 12 base pairs per complete turn the helix is considerably underwound. As observed with most oligodeoxyribonucleotides analysed by X-ray crystallography so far, the octamer displays reduced base pair tilt, increased rise per base pair and a more open major groove compared with canonical A-DNA. We propose that, based on these parameters, three A-helical sub-families may be defined; d(GGGATCCC) then is a representative of the class with intermediate tilt, rise, and major groove width.     

P-glycoprotein catalytic mechanism: studies of the ADP-vanadate inhibited state

Kinetics of inhibition of ATPase activity of pure mouse Mdr3 P-glycoprotein upon incubation with MgADP and vanadate were studied along with the trapping of [14C]ADP in presence of vanadate. The presence of verapamil strongly magnified both effects. Inhibition of ATPase was also increased by several other drugs known to bind to drug-binding sites. Inhibition by ADP-vanadate was slow and depended cooperatively on nucleotide binding. Stoichiometry of [14C]ADP trapping by vanadate was 1 mol/mol P-glycoprotein at full inhibition. Catalytic site mutants prevented [14C]ADP trapping, whereas interdomain signal communication mutants reduced it in approximate correlation with their effects upon drug stimulation of ATPase. In explanation of the results, we propose that a "closed conformation" involving dimerization and interdigitation of the two nucleotide-binding domains is necessary to allow inhibition by ADP-vanadate. The results suggest that such a conformation occurs naturally during ATP hydrolysis. It is proposed that in order for the catalytic transition state to form, the two nucleotide-binding domains dimerize to form an integrated single entity containing two bound ATP with just one of the two ATP being hydrolyzed per dimerization event.     

Membrane orientation of carboxyl-terminal half P-glycoprotein: topogenesis of transmembrane segments.

Multiple topological orientations of the carboxyl-terminal half of P-glycoprotein have been observed. One orientation is consistent with the hydropathy-predicted model and contains six transmembrane (TM)-spanning regions. In another orientation, the cytoplasmic-predicted loop between TM8 and TM9 is extracellular and glycosylated. In support of this "alternative" topology, TM8 was previously established to function as a signal-anchor sequence to insert with its amino-terminal end in the cytoplasm and the carboxyl-terminal end in the extracytoplasmic space. However, it is unclear how downstream TM segments fold in the membrane when TM8 functions as a signal-anchor sequence. Here, we created several chimeric Pgp molecules to examine the membrane insertion of TM segments 9 and 10 using a cell-free system. We found that TM9 functions as a stop-transfer sequence when following the signal-anchor sequence, TM8. However, the stop-transfer activity of TM9 depends on the presence of TM10. In the absence of TM10, TM9 partially translocated across the membrane into the endoplasmic reticulum lumen. In contrast, TM9 efficiently stopped the translocation event of the nascent chain in the presence of TM10. Our results suggest that the membrane insertion of TM8 and TM9 establishes the extracellular loop between TM8 and TM9. Formation of this loop apparently involves the interactions between Pgp TM segments, which facilitate proper folding of the Pgp carboxyl-terminal half.     

Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore

Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.     

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.     

Three-dimensional structure of the barley beta-D-glucan glucohydrolase in complex with a transition state mimic

Glucophenylimidazole (PheGlcIm), a tetrahydroimidazopyridine-type inhibitor and 4H3 conformer mimic of a glucoside, binds very tightly to a barley beta-d-glucan glucohydrolase, with a Ki constant of 2 x 10(-9) m and a DeltaG of 51 kJ mol(-1). PheGlcIm binds to the barley beta-d-glucan glucohydrolase approximately 2 x 10(5) times tighter than laminarin, which is the best non-synthetic ground-state substrate found so far for this enzyme, 10(6) times tighter than 4-nitrophenyl beta-d-glucopyranoside, and 2 x 10(7) tighter than glucose. The three-dimensional structure of the beta-d-glucan glucohydrolase with bound PheGlcIm indicates that the complex resembles a hypothetical transition state during the hydrolytic cycle, that the enzyme derives substrate binding energy from the "aglycone" portion of the ligand, and that it also reveals an anti-protonation trajectory for hydrolysis. Continuous electron densities at the 1.6 sigma level form between the three active site residues Asp95, His207, and Asp285, and the C6OH, C7OH, C8OH, and C9OH groups of PheGlcIm. These electron densities correspond to the most favorable interactions in the three-dimensional structure of the beta-d-glucan glucohydrolase-PheGlcIm complex and indicate atomic distances equal to or less than 2.55 A. The crystallographic data were corroborated with ab initio molecular orbital calculations. The data indicate that the 4E conformation of the glucose part of PheGlcIm is critical for tight binding and provide the first evidence for probable substrate distortion during catalysis by this enzyme.     

A coiled-coil structure of the alphaIIbbeta3 integrin transmembrane and cytoplasmic domains in its resting state

One of the hallmark features of the integrin receptors is the ability to transmit signals bidirectionally through the cell membrane. The transmembrane integrin domains are pivotal to the signaling events. An understanding of the signaling mechanism requires structural information. Here, we report a structural model of the transmembrane and part of the cytosolic domains of the alphaIIbbeta3 integrin in its resting state. The model was obtained computationally by a restrained conformational search of helix-helix interactions. It agrees with one published NMR structure of the cytoplasmic complex and can put many experimental findings on structural grounds. According to our model, integrins form an intricately designed coiled-coil structure in the resting state. The conserved Glycophorin A (GpA)-like sequence motif of the alpha, but not the beta, subunit, is in the interface of this model. Based on our calculations and other data, a signaling mechanism that involves a transient GpA-like structure is proposed.     

Oligomerization state and supramolecular structure of the HIV-1 Vpu protein transmembrane segment in phospholipid bilayers

HIV‐1 Vpu is an 81‐residue protein with a single N‐terminal transmembrane (TM) helical segment that is involved in the release of new virions from host cell membranes. Vpu and its TM segment form ion channels in phospholipid bilayers, presumably by oligomerization of TM helices into a pore‐like structure. We describe measurements that provide new constraints on the oligomerization state and supramolecular structure of residues 1–40 of Vpu (Vpu_1–40), including analytical ultracentrifugation measurements to investigate oligomerization in detergent micelles, photo‐induced crosslinking experiments to investigate oligomerization in bilayers, and solid‐state nuclear magnetic resonance measurements to obtain constraints on intermolecular contacts between and orientations of TM helices in bilayers. From these data, we develop molecular models for Vpu TM oligomers. The data indicate that a variety of oligomers coexist in phospholipid bilayers, so that a unique supramolecular structure can not be defined. Nonetheless, since oligomers of various sizes have similar intermolecular contacts and orientations, molecular models developed from our data are most likely representative of Vpu TM oligomers that exist in host cell membranes.     

Shape changes of giant liposomes induced by an asymmetric transmembrane distribution of phospholipids.

The influence of a phospholipid transmembrane redistribution on the shape of nonspherical flaccid vesicles was investigated at a fixed temperature by optical microscopy. In a first series of experiments, a transmembrane pH gradient was imposed on egg phosphatidylcholine (EPC)-egg phosphatidylglycerol (EPG) (100:1) giant vesicles. The delta pH induced an asymmetric distribution of EPG. Simultaneously, discoid vesicles were transformed into tubular or a series of connected small vesicles. The fraction of phospholipid transfer necessary for a shape change from discoid to two connected vesicles was of the order of 0.1% of the total phospholipids. Additional lipid redistribution was accompanied by a sequence of shape changes. In a second series of experiments, lyso phosphatidylcholine (L-PC) was added to, or subtracted from, the external leaflet of giant EPC vesicles. The addition of L-PC induced a change from discoid to a two-vesicle state without further evolution, suggesting that lipid transfer and lipid addition are not equivalent. L-PC depletion from the outer leaflet generated stomatocyte-like vesicles. Whenever possible, we have determined whether the giant vesicles undergoing shape changes were unilamellar or multilamellar by measuring the elastic area compressibility modulus, K, by the micropipette assay (Kwok and Evans, 1981). Shape transformations triggered by phospholipid modification of the most external bilayer were indeed influenced by the presence of other underlying membranes that played a role comparable to that of a passive cytoskeleton layer. It appears that in real cells, invaginations of the plasma membrane or budding of organelles could be triggered by a phospholipid transfer from one leaflet to the other caused, for instance, by the aminophospholipid translocase which is present in eukaryotic membranes.     

Three-dimensional structure of an invertebrate intercellular communicating junction

Gap junctions containing extensive, highly ordered crystalline arrays of hexagonally packed connexons have been isolated from the hepatopancreas of the arthropod, Homarus americanus (American lobster). The structure of such junctions has been studied to a resolution of approximately 25 A in three dimensions by electron microscopy of negatively stained specimens. The structure, which has the crystallographic symmetry of the two-sided plane group p6, reveals the connexon as an annular oligomer which projects approximately 30-45 A from the cytoplasmic surface. The stain-filled channel structure appears to be approximately 40-45 A wide in the extracellular region. Projection images of glucose-embedded specimens extend to a resolution of 10 A, and show a strong contrast from the connexon subunits. Overall the structure is quite similar to that of rat liver junctions, except that less stain is seen in the aqueous region of the gap and more surrounding the protrusions of the protein into the cytoplasm.     

Kinetics in the pre-steady state of the formation of cystines in ribonucleoside diphosphate reductase: evidence for an asymmetric complex

Two folded polypeptides, designated R1 and R2, respectively, combine in an as yet undefined stoichiometry to form ribonucleoside diphosphate reductase (ribonucleotide reductase) from Escherichia coli. Two pairs of cysteines in each R1 protomer have been implicated in the enzymatic mechanism. One pair, cysteines 225 and 462, is located in the active site of the enzyme and forms a cystine concomitant with the reduction of the ribonucleotide. The other pair, cysteines 754 and 759, is located near the carboxy terminus and is thought to reduce the cystine in the active site by disulfide interchange; either thioredoxin or glutaredoxin is then thought to reduce the cystine that results. Rapid quenching and site-directed immunochemistry have been used to follow the formation of the cystine in the active site and the peripheral cystine simultaneously during the pre-steady state. Prereduced R1 dimer of ribonucleoside diphosphate reductase, in the presence of ATP and CDP, was mixed with R2 dimer in an apparatus for quench flow. The reaction was quenched with a solution of acetic acid and N-ethylmaleimide, the protein was then precipitated with trichloroacetic acid, and the precipitate was separated into two portions. The percent of the cystine in the active site in one of the portions was determined as described previously [Erickson, H. K. (2000) Biochemistry 39, 9241-9250]. A similar method was employed to determine the percent of the peripheral cystine in the other portion of the precipitate. It was found that while the formation of both of these cystines was initiated by the addition of R2 dimer, presumably as products of the reduction of CDP, the peripheral cystine appeared to form more rapidly and in a higher yield than the cystine in the active site. These results demonstrate that the formation of the cystine between cysteines 754 and 759 of ribonucleotide reductase from E. coli is kinetically competent. A mechanism consistent with the prior formation of the cystine between cysteine 225 and cystene 462 as well as the kinetics for the formation of each cystine with time is presented. Because twice as much of the peripheral cystine than cystine in the active site had formed during the pre-steady state, it follows that the enzymatically competent complex between R1 dimers and R2 dimers cannot be symmetric.     

Three-dimensional reconstruction of dynamin in the constricted state

Members of the dynamin family of GTPases have unique structural properties that might reveal a general mechanochemical basis for membrane constriction. Receptor-mediated endocytosis, caveolae internalization and certain trafficking events in the Golgi all require dynamin for vesiculation. The dynamin-related protein Drp1 (Dlp1) has been implicated in mitochondria fission and a plant dynamin-like protein phragmoplastin is involved in the vesicular events leading to cell wall formation. A common theme among these proteins is their ability to self-assemble into spirals and their localization to areas of membrane fission. Here we present the first three-dimensional structure of dynamin at a resolution of approximately 20 A, determined from cryo-electron micrographs of tubular crystals in the constricted state. The map reveals a T-shaped dimer consisting of three prominent densities: leg, stalk and head. The structure suggests that the dense stalk and head regions rearrange when GTP is added, a rearrangement that generates a force on the underlying lipid bilayer and thereby leads to membrane constriction. These results indicate that dynamin is a force-generating 'contrictase'.