Inward-facing conformation of a multidrug resistanceMATE family transporterSandra Zakrzewskaa, Ahmad Reza Mehdipourb, Viveka Nand Malviyaa, Tsuyoshi Nonakaa, Juergen Koepkea,Cornelia Muenkea, Winfried Hausnerc, Gerhard Hummerb,d, Schara Safariana,1, and Hartmut Michela,1

aDepartment of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany; bDepartment of TheoreticalBiophysics, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany; cFaculty of Microbiology, University of Regensburg, 93053 Regensburg,Germany; and dInstitute of Biophysics, Goethe University, 60438 Frankfurt am Main, Germany

Contributed by Hartmut Michel, May 2, 2019 (sent for review March 12, 2019; reviewed by Raimund Dutzler and José D. Faraldo-Gómez)

Multidrug and toxic compound extrusion (MATE) transportersmediate excretion of xenobiotics and toxic metabolites, therebyconferring multidrug resistance in bacterial pathogens and cancercells. Structural information on the alternate conformational statesand knowledge of the detailedmechanism ofMATE transport are ofgreat importance for drug development. However, the structures ofMATE transporters are only known in V-shaped outward-facingconformations. Here, we present the crystal structure of a MATEtransporter from Pyrococcus furiosus (PfMATE) in the long-sought-after inward-facing state, which was obtained after crystallizationin the presence of native lipids. Transition from the outward-facingstate to the inward-facing state involves rigid body movements oftransmembrane helices (TMs) 2–6 and 8–12 to form an inverted V,facilitated by a loose binding of TM1 and TM7 to their respectivebundles and their conformational flexibility. The inward-facing struc-ture of PfMATE in combinationwith the outward-facing one supportsan alternating access mechanism for the MATE family transporters.

multidrug resistance | membrane protein structure | MATE transporter |inward-facing conformation | lipids

The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP)transporter superfamily is mainly divided into four major dis-tantly related families: multidrug and toxic compound extrusion(MATE), polysaccharide transporter (PST), oligosaccharidyl-lipidflippase (OLF), and mouse virulence factor (MVF) (1). Amongthese, the MATE transporters are most ubiquitous, being presentin all domains of life (2). As secondary active transporters, theyutilize transmembrane electrochemical ion gradients (Na+ or H+)(3–6) to drive the export of xenobiotics or cytotoxic metabolicwaste products with specificity mainly for organic cations. MATEtransporters confer resistance, for example, to fluoroquinolones,aminoglycoside antibiotics, and anticancer chemotherapeuticalagents (4, 6), thus serving as promising drug targets in the fightagainst multidrug resistance. Based on their amino acid sequencesimilarity, the MATE family members are classified into theNorM, the DNA damage-inducible protein F (DinF), and theeukaryotic subfamilies (2). In recent years, the crystal structures ofrepresentative members of all three subfamilies have been pub-lished (7–15). Since all of them represent only an outward-facingstate, a detailed understanding of the complete transport cycle hasremained elusive.This work is focused on the DinF subfamily member, PfMATE

(UniProtKB entry: Q8U2X0), which is one of the four annotatedMATE transporters of the hyperthermophilic archaeonPyrococcus furiosus. We could produce this protein in recombinantform in Escherichia coli, crystallize it, and determine the structurein an outward-facing conformation at the high resolution of 2.35 Å[Protein Data Bank (PDB) ID: 4MLB] (SI Appendix, Fig. S1). Thestructure of PfMATE is similar to NorM_VC from Vibrio choleraeand NorM_NG from Neisseria gonorrhoeae (7, 10), except for theelectrostatic character of the substrate-binding cavity. TM1–TM6and TM7–TM12 form helical bundles, called the N-lobe and C-lobe, respectively, creating a V-shaped cleft open to the extra-

cellular side. However, opposite to the classical MATE trans-porters, the large central cavity of PfMATE is strongly positivelycharged (SI Appendix, Fig. S2A), which may be suited for ac-commodation of negatively charged lipophilic compounds, par-ticularly lipids. Considering deviations between archaeal andbacterial lipids, we conducted crystallization experiments in thepresence of native lipids extracted from P. furiosus. This approachled to the crystallization and structure determination of PfMATEin an inward-facing conformation, hinting at a crucial role of en-dogenous lipids in the functionality of this transporter. A loosebinding of TM1 and TM7 to their respective lobes and theirconformational flexibility appear to be key elements for the tran-sition between the outward- and inward-facing conformations,including rigid body movements of TM2–TM6 and TM8–TM12.To investigate the interactions between PfMATE and its nativelipids, we also performed molecular dynamics (MD) simulations inan archaeal-type lipid bilayer. Furthermore, the combination ofour data from single-wavelength anomalous dispersion (SAD)

Significance

Active efflux of drugs and toxic compounds carried out by mul-tidrug and toxic compound extrusion (MATE) family transportersis one of the strategies developed by bacterial pathogens toconfer multidrug resistance. To elucidate the underlying steps ofthe MATE transport cycle, structures of distinct intermediates arerequired; however, only structures in outward-facing conforma-tions have been available. Our approach of using native lipidsallowed trapping and visualization of the MATE transporter fromPyrococcus furiosus in an inward-facing conformation. This workhighlights the importance of native lipids and opens an alternativeview on the function and mechanism of action for the MATEfamily transporters.

Author contributions: S.Z. prepared samples, and designed and performed research; S.Z.and S.S. implemented X-ray data acquisition and solved the 6GWH and 6FHZ structures;S.Z. performed structure refinement; A.R.M. performed MD simulations; T.N. solved theoriginal 6HFB and 4MLB structures; S.Z. and J.K. refined the 6HFB and 4MLB structures;C.M. performed cloning, optimization of gene expression, purification, and crystallizationleading to the 6HFB and 4MLB structure determination; V.N.M. performed the functionalcharacterization of PfMATE; W.H. assisted with the cultivation of P. furiosus; S.Z., A.R.M.,and S.S. prepared figures; A.R.M. prepared videos; S.Z., A.R.M., and S.S. wrote the man-uscript with contributions from G.H. and H.M.; H.M. suggested experiments; G.H. andH.M. supervised the project.

Reviewers: R.D., University of Zurich; and J.D.F.-G., National Institutes of Health.

The authors declare no conflict of interest.

Published under the PNAS license.

Data deposition: The structure factors and coordinates for the inward- and outward-facing states obtained by the lipidic cubic phase method were deposited in the ProteinData Bank (PDB), www.pdb.org (PDB ID codes 6FHZ and 6GWH, respectively). The crys-tallographic data corresponding to the outward-facing structures obtained by the vapordiffusion method were deposited in the PDB (PDB ID codes 6HFB and 4MLB).1To whom correspondence may be addressed. Email: schara.safarian@biophys.mpg.de orhartmut.michel@biophys.mpg.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1904210116/-/DCSupplemental.

Published online June 3, 2019.

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Fig. 1. Crystal structures of PfMATE in two distinct conformations. (A) Side view of PfMATE representing outward-facing (OFC) and inward-facing (IFC)conformations. Ribbon models of the N-lobe (TM1–TM6) and the C-lobe (TM7–TM12) are shown in pink and blue, respectively. The dashed lines representthe borders of the lipid bilayer. (B, Left) Structural superposition of the N-lobes and C-lobes of two states, indicating a substantial alteration of TM1 in theN-lobe and TM7 in the C-lobe, whereas the remaining helices undergo a rigid body movement. (B, Right) Structural alignment of the N-lobe and C-lobe ofthe IFC shows their symmetrical arrangement. (C ) Surface representation of the OFC and IFC showing the central binding cavity from the top and incross-section.

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experiments using anomalous signals of Cs+ with the results ofMD simulations indicates the presence of a monovalent cation-binding site at Asp41. Based on these data, we could establish aframework of the structural changes that occur during the tran-sition between the inward- and outward-facing conformations ofthe MATE transporter.

ResultsCrystal Structure of PfMATE in the Inward-Facing Conformation. OurPfMATE structure in an outward-facing state (PDB ID: 4MLB)revealed that the V-shaped central vestibule contains four posi-tively charged residues (Arg83, Arg161, Arg284, and Arg402).Furthermore, the interior of the central cavity is lined with acluster of methionine residues (Met27, Met31, Met64, Met173,Met256, Met260, Met287, and Met381), creating a hydrophobicenvironment around the center of the membrane. Remarkably,the positively charged cavity of PfMATE shows a reverse polaritycompared with the canonical MATE transporters, such asNorM_NG and NorM_VC, as well as the eukaryotic MATEs.Among the MOP superfamily, the PfMATE vestibule rather re-sembles that of the MurJ lipid II flippase from the MVF family,which is predominantly cationic (16) (SI Appendix, Fig. S2B).Crystallization of PfMATE in the presence of endogenous

lipids extracted from the source organism P. furiosus, but notfrom E. coli, led to structure determination of PfMATE in aninward-facing conformation with the central cavity accessible forsubstrates from the intracellular side (Fig. 1A and SI Appendix,Table S2). The prominent feature of this conformation is thesignificantly bent TM1, which undergoes a notable secondarystructure rearrangement (SI Appendix, Fig. S3). A partial un-winding of this helix hints at a high degree of flexibility, sug-gesting a role in the conformational switch between outward-and inward-oriented states. A structural superposition betweenthe N-lobe and C-lobe of the inward-facing structure [global Cαroot-mean-square deviation (RMSD) = 2.58 Å] demonstratesthat the internal twofold symmetry between these two domainsremains preserved (Fig. 1B). The conformational rearrange-ment of TM7 of the C-lobe is similar to that of its symmetry-related counterpart TM1 of the N-lobe, which undergoes sub-stantial bending and is located in close proximity to the C-lobehelices (Fig. 1B). These structural alterations cause the in-tracellular opening to be significantly wider (about 26 Å) thanthe extracellular one (about 18 Å) in the respective accessstates (Fig. 1C).

Structural Basis of the Extracellular and Intracellular Barrier Formation.Fundamentally, secondary active transporters use electrochemicalgradients of either protons or sodium ions to enable the uphillmovement of substrates across the membrane. According to thebasic principles of the alternating access model (17, 18), MATEtransporters sequentially expose their substrate-binding cavity toeither side of the membrane during a transport cycle by internal orexternal barrier formation. These barriers restrict the accessibilityof the substrate- and ion-binding sites to one respective sur-rounding at a time. A structural alignment of the N-lobes (CαRMSD = 2.02 Å) and C-lobes (Cα RMSD = 1.84 Å) of thePfMATE outward- and inward-facing conformations reveals thatthese barriers are formed by local and global conformationalchanges, including rigid body movements of the N- and C-lobes, ahinge-like motion of the N-terminal segment of TM1, and un-winding of TM1 halfway across the membrane. Alternating accessto the substrate-binding site appears to be enabled by significantmovements of TM1 and TM7, while the remaining TMs of the N-lobe (TM 2–TM6) and the C-lobe (TM8–TM12) stay together andundergo a rigid body movement.In the inward-facing conformation, the extracellular barrier is

mediated predominantly by hydrophobic and aromatic interac-tions between residues located at the extracellular apex of the

lobes. This tightly closed barrier with a thickness of about 10 Åinvolves hydrophobic interactions between Pro50 (TM2), Leu53(TM2), Ala54 (TM2), Ala277 (TM8), Ala355 (loop connectingTM9 and TM10), Val357 (TM10), and Ile358 (TM10) (SI Ap-pendix, Fig. S4A). The van der Waals interactions between Phe349(TM9) and Ile43 (TM1), Gly49 (TM1), and Ile285 (TM8) serve askey components for the formation of the extracellular barrier,which blocks access to the substrate-binding site from the extra-cellular side of the membrane. The displacement of TM7 andTM8 at the extracellular apex of the C-lobe from their positions inthe outward-facing structure, closely approaching the helices ofthe N-lobe, is probably due to the hydrophobic interactions be-tween the residues Leu58 (TM2), Val62 (TM2), Leu122 (TM3),Met126 (TM3), Ala128 (loop between TM3 and TM4), Phe261(TM7), Ile268 (TM7), and Val276 (TM8) (SI Appendix, Fig. S4B).The hydrophobic interaction between Met64 (TM2) and Met260(TM7) is most likely associated with the closure of the extracel-lular gate and the formation of a hydrophobic groove within the C-lobe. A network of interactions between several residues from theC-lobe creates this groove: Leu263 (TM7), Phe279 (TM8), Trp283(TM8), Met287 (TM8), Leu369 (TM10), Phe372 (TM10), Ala411(TM11), Val425 (TM12), Trp426 (TM12), and Ile429 (TM12).Additionally, the extracellular barrier is stabilized by a hydrogenbond between the carbonyl oxygen atom of Ile43 (TM1), the side-chain OH group of Tyr351 (loop between TM9 and TM10), andthe side-chain OH group of Ser46 (TM1).When PfMATE switches from the inward-facing state to the

outward-facing state, the interaction network contributing to theclosure of the extracellular barrier has to be disrupted. Forma-tion of the intracellular barrier obstructs solvent accessibilityfrom the cytoplasm, while the extracellular barrier opens (MovieS1). Thus, the substrate- and ion-binding sites are accessible viaan aqueous cavity, which is exposed to the extracellular side ofthe membrane. The thicker barrier between the substrate andthe cytoplasm (about 14 Å) (Fig. 2A) may be correlated with thesubstantial movement of the intracellular half of TM1. Consis-tent with the positive inside rule (19), the intracellular side of thePfMATE contains more positively charged residues comparedwith the extracellular barrier. Therefore, not only hydrophobicinteractions, which are predominant in the extracellular barrier,but also ionic interactions, such as the salt bridge formed be-tween the side chains of Arg244 (TM7) and Glu393 (TM11),strengthen the intracellular barrier. The ionic interactions be-tween the side chains of Glu310 (TM8), Arg13 (TM1), andArg88 (TM2) are likely to be the driving factor for the movementof TM8 and TM9 on the intracellular side, thereby closing theintracellular pathway (SI Appendix, Fig. S4C). In the outward-facing structure, the side chain of Arg244 (TM7) is flipped awayfrom Asp241 (TM7) and forms an ionic interaction with the sidechain of Glu393 (TM11), bringing TM7 and TM11 together.Interestingly, the side chains of Arg13 (TM1) and Arg88 (TM2)are stacked in an antiparallel manner at the apex of the in-tracellular barrier. This coordination of two positively chargedresidues may imply the existence of a repulsive electrostatic gate,which could preclude a flux of protons and other cations, therebyforming a tight barrier preventing ion leakage during the confor-mational transition. The hydrophobic interaction between Val9(TM1) and Ile85 (TM2) is another major contributor in the for-mation of the intracellular barrier, which stabilizes the position ofthe short helical stretch of the N terminus in the outward-facingstructure. An aqueous path to the intracellular side is also closedthrough a hydrophobic interaction network between Ala82, Gly86(TM2), and Ala306 (TM8). The outward- and inward-facingconformations of PfMATE illustrate how these two distinct bar-riers are formed by a network of mostly hydrophobic residues atthe closely interacting lobe interfaces in each state.

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Structural and Functional Implications of TM1 Bending. In the pre-viously proposed model of the PfMATE transport, the highlyconserved Asp41 constitutes the key residue associated withproton coupling (14). It has been proposed that Asp41 isdeprotonated at high pH and that its protonation induces theconformational change of TM1 from the straight state to thebent state. This conclusion was based on two PfMATE crystalstructures obtained at high pH (7.0–8.0) and low pH (6.0–6.5),respectively. The aforementioned interpretation raised somecriticism, leading to the hypothesis about TM1 bending to berather affected by interactions with exogenous lipids (mono-olein) present in the crystallization conditions as described pre-viously (13, 20). This discrepancy motivated us to evaluate therelationship between pH and structural alteration of TM1.Therefore, we determined outward-facing structures of PfMATEfrom crystals generated at low pH (5.0 and 6.5) by both vapordiffusion and lipidic cubic phase (LCP) methods (SI Appendix,Fig. S7). A comparison between these structures and the pre-viously presented high pH outward-facing structure (PDB ID:

3VVN) reveals that TM1 remains straight irrespective of thepH of the crystallization media; there are no major structuraldifferences, as reflected by a very low Cα RMSD = 0.86 Å ofthe structure generated in the presence of the exogenous lipids(PDB ID: 6GWH); 0.69 Å, 0.71 Å, 1.08 Å, and 0.64 Å of chainsA, B, C, and D, respectively, (PDB ID: 4MLB); and 0.74 Å,0.77 Å, 1.10 Å, and 0.65 Å of chains A, B, C, and D, re-spectively, (PDB ID: 6HFB) of the structures obtained in theabsence of monoolein molecules. Hence, our results are con-sistent with the notion that the conformational rearrangementsof helix TM1 are most likely independent of pH. In recentlypublished MD simulations of PfMATE with bent TM1 (PDBID: 3VVO) in the outward-facing conformation, replacing awater molecule near Asp41 by sodium produced a stablestructure (21).Nevertheless, besides the inversion of the access state, the

largest structural difference between the outward- and inward-facing conformations is found within TM1. Notably, a pivot point atGly30 allows the large hinge-like movement within the N-terminal

Fig. 2. Sodium ion-binding site in PfMATE and water accessibility. (A) Water occupancy of the binding-site cavity (blue surface) in simulations of inward-facingand outward-facing conformations. Water access to the cavity is restricted to the inside and outside, respectively. (B) Distance between the sodium ion-bindingsite (carboxyl oxygen atoms of Asp41) and a Na+ that binds spontaneously during MD simulations of the outward-facing conformation (OFC) state (Top) and thatremained bound in the inward-facing conformation (IFC) state (Bottom). Sodium ion-binding site in the IFC state, based on the crystal structure (PDB ID code 6FHZ)(C) and the representative MD snapshot (D). The residues labeled and shown by a stick model coordinate the bound Na+, depicted as an orange sphere. Thepurple mesh represents the Fo-Fc electron density peak, which is tentatively assigned to the Na+ ion. The sodium ion-binding site in the OFC state, based on thecrystal structure (PDB ID code 6HFB) (E) and an MD snapshot of the fully coordinated sodium ion (taken from the simulation at 700 ns) (F). The coordinatingresidues are shown in stick representation, whereas the blue mesh shows the anomalous peak for Cs+.

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segment of TM1 to adopt the inward-facing conformation. Theunfolded intramembrane region in TM1 exhibits remarkableflexibility, suitable for coordination of substrates or ions. Duringthe transition from the outward- to inward-facing state, two seg-ments of TM1, one from Ala17 to Ala31 and the second fromSer32 to Val45, tilt relative to each other. This tilting can bedecomposed into two movements described by the following an-gles: The first angle defines the movement of TM1 as the gate-keeper of the lateral opening in the Y/Z plane (about 27°), whilethe second angle describes the pronounced movement in the X/Zplane (about 42°), showing that the intramembrane half ofTM1 also acts as a plug to seal the lateral opening in the outward-facing conformation. In the inward-facing structure, the side chainof Phe60 (TM2) is flipped toward Val56 (TM2), also creating ahydrophobic contact with Gly42 (TM1), which leads to a helicalkink. In the outward-facing structure, these residues are shiftedapart and TM1 remains straight. The bending of TM1 in theinward-facing state is further facilitated by side-chain interactionsbetween Asn154 (TM10) and Gln34 (TM1). A hydrogen bondbetween the side-chain carboxyl group of Asp41 (TM1) and theside-chain hydroxyl group of Tyr139 (TM4), as well as a hydro-phobic interaction between Pro26 (TM1) and Ala166 (TM5), ismost likely involved in the formation of the kinked state of TM1.The substantial movements of TM1 toward the C-lobe in theinward-facing state appear to contribute also to the widened in-tracellular opening compared with the extracellular state (Fig.1C). The structural alteration of TM1 in the inward-facing struc-ture implies a highly dynamic nature and flexibility of this helix,indicative of a functional role in substrate and ion gating.

Ion-Binding Sites. MATE proteins are secondary active trans-porters harnessing the energy from an electrochemical trans-membrane ion gradient (Na+ or H+) to enable substrate transport.The ion-binding site in the outward-facing conformation ofPfMATE is located near Asp41, Asn180, Asp184, and Thr202close to the surface of the internal cavity aqueous path leading tothe ion-binding site in the outward-facing structure, and it isplaced in a water-accessible surrounding, whereas in the inward-facing conformation, it appears to be occluded. Sodium ions andwater molecules are not straightforwardly distinguishable throughX-ray crystallography. Tanaka et al. (14) interpreted an electrondensity peak near this site as a bound water molecule in theoutward-facing state (PDB ID: 3VVO and 3VVN). A highlysimilar ion-binding site formed by Asp35, Asn174, Asp178,Ala192, and Thr196 (corresponding to the homologous residuesAsp41, Asn180, Asp184, Ala198, and Thr202 of PfMATE) wasidentified in the X-ray structure of another MATE transporterfrom V. cholerae, VcmN (8). The electron density signal at this sitewas also assigned to a water molecule by analogy with the afore-mentioned studies (14). Our CsCl heavy atom derivative dataset ofthe outward-facing structure (PDB ID: 6HFB) shows an anoma-lous difference electron density for a Cs+, adjacent to the carboxyloxygen atom Oδ1 of Asp41 (Fig. 2E). Presumably, due to thelarger size, cesium ions do not occupy exactly the same position asa Na+. The anomalous signal from a Cs+ demonstrates the neg-ative electrostatic potential of this site, which is suited for bindingmonovalent cations. As shown in the recent MD simulations, theoutward-facing bent state of PfMATE (PDB ID: 3VVO) reveals ahighly coordinated Na+-binding site (21). This sodium ion-occluded bent state appears to be an intermediate in the trans-port cycle stabilized by ion binding before transition into theinward-facing state. Our MD simulations of the outward-facingstructure showed spontaneous Na+ binding to the Cs+ site ofthe crystal structure (Fig. 2B). After ion recognition, the bindingsite did not immediately close and the sodium ion formed fourinteractions with the protein (two with Asp41 and one each withAsn180 and Thr202), but retained two water molecules. Laterduring the simulation, the missing protein interactions with

Asp184 and Ala198 formed, replacing the two water molecules,resulting in a coordination structure as in the inward-facing stateand in the simulation of the outward-facing structure (PDB ID:3VVO) (21) (Fig. 2F). However, the interactions of bound Na+

with Asp184 and Ala198 are transient. Interestingly, in the fullycoordinated state, TM1 is slightly bent, adopting an intermediateconformation between those of 3VVO and 3VVN.A prominent spherical electron density signal was also ob-

served in the Fo-Fc map of the inward-facing structure in veryclose proximity to Asp41 (Fig. 2C), which is consistent with acoordination number of six. This observation raises the possi-bility that the electron density in the center is caused by thepresence of a Na+ ion. Also, the coordination is highly similar tothat of Na+ sites in other proteins of known structure (22, 23).The distances between the sodium ion and oxygen atoms of thecoordinating residues in this structure [Oδ1 of Asp41 (2.42 Å),Oδ1 of Asn180 (2.45 Å), Oδ1 of Asp184 (2.60 Å), carbonyl ox-ygen atom of Ala198 (2.08 Å), and Oγ of Thr202 (2.38 Å)] alsoindicate a Na+-bound state. The presence of a monovalent cation-binding site in the N-lobe of PfMATE was supported by our MDsimulations, which showed a Na+-binding site near Asp41 (pKa =5.1) and Asp184 (pKa > 14) in the outward-facing structure. Thecalculated pKa values for the inward-facing structure stronglyimply that in the absence of sodium ions, both Asp41 (pKa =13.7) and Asp184 (pKa > 14) are protonated, while in thepresence of sodium ions, Asp41 (pKa = 0) is most likely charged(Fig. 2D). Results obtained from the MD simulations in thepresence of sodium ions indicate a well-coordinated binding site,while in the absence of sodium ions, the protonation of bothresidues, Asp41 and Asp184, is essential for the stability of thebinding site (Fig. 2 B and D). In light of the high calculated pKaof Asp41 in the inward-facing structure (pKa = 13.7 in the ab-sence of sodium ions), Na+ release could be coupled toAsp41 protonation. Structuring of the unwound TM1 segmentcould be another relevant factor. The structural data and theresults of the MD simulations imply a sodium ion-dependenttransport mechanism of PfMATE. However, we cannot ex-clude a dual specificity for sodium ions and protons, which wasproposed in the recent publications demonstrating a dual ioncoupling behavior for NorM_VC (24, 25).Surprisingly, during the MD runs, we also observed sponta-

neous binding of chloride ions into the cavity in the inward-facing structure (SI Appendix, Fig. S8). The chloride interactswith Arg284, Thr35, Asn38, Trp283, and Met287. Interestingly, aCl− has also been observed near Arg24 and Arg255 in the MurJinward-facing structure (16), which corresponds to a similarposition of PfMATE after superposition of both structures. Wealso observed Cl− binding into the outward-facing structure in adifferent site near Arg402. In the archaeal lipid simulation, thelipid head group subsequently replaced the Cl− ion. By contrast,in the bacterial lipid simulation, after 1.2 μs, both a lipid and achloride ion were bound. Currently, the functional importance ofthe chloride ion for PfMATE remains unclear. We cannot ex-clude that the bound chloride is released to the external sideduring the transition to the outward-facing state in such a waythat MATE (or, in general, MOP) transporters are actuallychloride/sodium ion antiporters. The cotransport of a chlorideion would be energetically beneficial by contributing drivingforce to the transport.

Interaction of Archaeal Lipids with PfMATE. Lengthy electron den-sities in the map of the inward-facing conformation observed inproximity to the intramembrane region of TM1 and helicesTM8 and TM9 may originate from the lipid molecules. Since, atthe present resolution, these densities cannot be unambiguouslyassigned to lipids, they were not included during the structurerefinement. It is commonly known that lipids are very often poorlyresolved and/or difficult to distinguish from other molecules like

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detergents. The local environment for each lipid can influence theconformation of the lipid tails, their mobility, and disorder, lead-ing to variable conformations. Due to their flexibility or multiplealternative conformations, lipids can result in weak signals in theelectron density maps. Therefore, structural information on thelipid–protein interaction is difficult to obtain. We cannot empiricallyidentify a specific lipid-binding site and figure out how exactly theaddition of the native lipids leads to crystallization of this protein inthe inward-facing state. Nevertheless, having established that nativelipids act like conformational modulators of PfMATE, we carriedout all-atom MD simulations of this protein embedded in a lipidbilayer of archaeal lipids to assess the protein–lipid interplay.P. furiosus is a strictly anaerobic and hyperthermophilic

archaeon (26). To cope with the elevated temperature, pressure,and lack of oxygen, thermophilic archaea have evolved certainmechanisms, which allow surviving under extreme conditions.One of the fundamental thermal adaptations is the membranelipid composition (27). Our mass spectrometric analysis revealsthat P. furiosus membranes contain diphytanyl phosphati-dyl inositol (DPI), diphytanyl phosphatidyl N-acetyl hexose,diphytanyl phosphatidyl glycerol (DPG), diphytanyl phosphatidicacid (DPA), and isoprenoidal glycerol dialkyl glycerol tetraetherlipids. Based on these results, we performed MD simulations forthe interaction studies of PfMATE and lipids under native-likeconditions at an elevated temperature (100 °C) (Movie S2).PfMATE was embedded in an archaeal lipid bilayer, consistingof DPI, DPG, and DPA lipid species (ratio of 45:20:35%). OurMD simulation results for the outward-facing state show lipidmolecules moving into the cavity and being accommodated bythe positively charged pocket. Interestingly, during severaldifferent MD simulation runs, we observed a completereorientation of negatively charged lipids (mostly PG species)inside the cavity, with the head group moiety pointing toward thebottom of the cavity (Fig. 3). In these simulations, the lipid en-tered the cavity from the outer leaflet. Initially, the lipid headgroup intruded into the cavity several times and then moved backand interacted mainly with Arg284 and Trp283; subsequently, apredominantly electrostatic interaction between the head groupand Arg402 resulted in a further movement of the head groupinside the cavity, which finally triggered the full reorientation ofthe lipid. After the reorientation, the head group interacted with

several residues at the bottom of the cavity, including Arg161,Thr399, Arg402, Asn253, and Gln387. Furthermore, a clusterof methionine residues, including Met31, Met64, Met256,Met260, and Met287, coordinated the aliphatic tails of thelipid. The lipid remained bound into the cavity for the rest ofthe simulation (up to 1.5 μs), with its headgroup stabilized byhydrogen bonds and electrostatic interactions and its tail sta-bilized by abundant hydrophobic interactions with the proteincavity. Lipid binding was observed only for the outward-facingstructure. In the inward-facing structure, closed lateral gatesblock lipid access to the cavity. Interestingly, we observed thesimultaneous lipid and sodium ion binding in the outward-facing state. Our results demonstrate the existence of a path-way for lipid access into the central cavity of PfMATE and thepossibility of lipid flipping.

DiscussionWe present here the inward-facing structure of a MATE familytransporter, namely, PfMATE, from P. furiosus. This structurehas to be expected to possess a high-affinity substrate-bindingstate that is not only essential from the physiological perspec-tive, but also may serve for the structure-aided design of inhib-itors. Together with the MD simulations, our structural dataimply lipid-specific regulation of conformational rearrangementsduring the alternating access mechanism of action of this trans-porter. Tanaka et al. (14) reported two outward-open structuresof PfMATE and convincingly presented data that PfMATEconfers norfloxacin resistance to the E. coli BW25113 ΔacrBstrain. Interestingly, the lipid or monoolein molecules were ob-served in the straight and bent outward-facing structures ofPfMATE (3VVO and 3VVN, respectively). The binding site oftwo monoolein molecules observed exclusively in the N-lobe oftheir straight structure (3VVN) corresponds to the drug recog-nition site in the Br-norfloxacin–bound structure (3VVP). Theauthors suggested that these molecules may mimic the hydro-phobic substrates. Based on our functional studies, the minimuminhibitory concentration (MIC) values were determined for severalantimicrobial compounds, including antibiotics and DNA-bindingdyes in the E. coli KAM32 strain (ΔacrB, ΔydhE, and hsdΔ5) andthree variants of the BW25113 strain, namely, BW25113A (ΔacrAB,ΔtolC, ΔydhE, ΔmdfA, and ΔemrE), BW25113B (ΔacrAB, ΔydhE,

Fig. 3. Spontaneous binding and reorientation of an archaeal lipid in MD simulations of PfMATE. (Left) Side view of the representative MD snapshotshowing the interaction of PfMATE (ribbons; N-lobe in pink and C-lobe in blue) with the archaeal-type DPG lipid (stick model) that entered the cavity from theoutside leaflet at the top and spontaneously flipped its orientation. (Right) Zoom-in view highlights residues contacting the lipid. MD snapshots have beentaken from the end of the 1.5-μs simulation.

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and ΔemrE), and BW25113C (ΔacrB) (SI Appendix, Fig. S5 andTable S1). The MIC values for different compounds were thesame for the test cells containing the plasmid with the gene codingfor PfMATE (pBAD-PfMATE) and the control cells containingthe empty vector (pBAD). Overexpression of PfMATE did notprovide any additional resistance against any of the tested com-pounds, implying that this transporter may be more specializedthan the classical MATE family members. Importantly, only the E.coli C43(DE3) ΔacrAB strain containing pBAD-PfMATE showedsome increased norfloxacin resistance compared with the cellswith the empty vector; however, the difference is not as significantcompared with the cells containing the gene coding for the MATEtransporter NorM_VP from Vibrio parahaemolyticus used as apositive control (SI Appendix, Fig. S6). Our results are also similarto those of functional studies on ClbM transporter from theMATE family, which did not provide resistance to ethidium bro-mide in E. coli KAM32 cells; however, a resistance was observedin E. coli C43(DE3) ΔacrAB cells. Despite the high structuralresemblance to the other MATE family transporters, particularlyPfMATE, ClbM has an additional unique function of pre-colibactin transport (12). Therefore, we cannot exclude that theprimary function of PfMATE is lipid transport. The interaction oflipid molecules with PfMATE also opens an alternative view onthe mechanism of action for the MATE family members, in whichspecific lipid species could either modulate the transition betweenthe outward- and inward-facing state or mimic the substrates forthese transporters. It is noteworthy that Martens et al. (28) re-cently proposed a model for the lipid-induced conformationalequilibrium of secondary active transporters based on hydrogen/deuterium exchange mass spectrometry and MD simulation ex-periments. This work particularly described how lipid binding toconserved networks of charged residues induces the conforma-tional transition toward an inward-facing conformation. Our re-sults comprise fundaments for future structural and functionalstudies of PfMATE modulation by different lipid species.Taken together, PfMATE is now the only transporter from the

MOP superfamily for which structures are available in an outward-facing as well as inward-facing conformation. The insights from ourstructural and computational studies thus pave the way toward abetter understanding of the underlying steps of the PfMATEtransport cycle and strongly favor the conclusion that native lipidsfrom the corresponding source organism and their specific inter-actions with protein may be of physiological relevance and essentialfor function and conformational regulation of MATE transportersin general.

MethodsGene Cloning and Mutagenesis. Genomic DNA of P. furiosus Vc 1 was obtainedfrom Michael Thomm and Harald Huber, Faculty of Microbiology, Universityof Regensburg. The target gene PfMATE (UniProtKB entry Q8U2X0) wascloned into the pBAD-A2 vector, a modification of the pBAD/HisA vector(Invitrogen) encoding a tobacco etch virus protease cleavage site and a 10×-His tag at the C terminus (29). Twenty-six variants of the wild-type proteinwere prepared by introducing cysteine residues at different positions,namely, E51C, V62C, L69C, K90C, E91C, N95C, V109C, S125C, M126C, L144C,M170C, S177C, D184C, S235C, E273C, E310C, T318C, I324C, E353C, A411C,V435C, I438C, M446C, K90C/E91C, M170C/T318C, and N95C/M170C/S235C/T318C. Four different types of 13 methionine residue-substituted variantswere also prepared (11ML/M170C/M446C, 12ML/M170C, 12ML/M446C, and13ML), in which 11 methionine residues (Met-27, Met-28, Met-31, Met-126,Met-173, Met-256, Met-260, Met-380, Met-381, Met-385, and Met-434) weresubstituted by leucine residues and M170 and M446 were substituted byeither cysteine or leucine residues. These constructs were prepared to obtainmercury and selenomethionine derivatives.

PfMATE Production and Purification.Inward- and outward-facing states (PDB ID codes 6FHZ and 6GWH). The geneencoding PfMATE was expressed in E. coli TOP10 cells (Invitrogen). Thecultures were grown in lysogeny broth (LB) medium supplemented with 50μg/mL carbenicillin and incubated with shaking at 37 °C. Once an optical

density at 600 nm (OD600) reached 0.6–0.8, the gene expression was inducedby addition of L-arabinose to a final concentration of 0.02% (wt/vol). After 3 hof incubation at 37 °C, cells were harvested by centrifugation at 4 °C for15 min. Bacterial cell pellets were resuspended in a fivefold excess (vol/wt) oflysis buffer composed of 20 mM Hepes-NaOH (pH 8.0), 300 mM NaCl, 5 mMMgCl2, a pinch of DNaseI, and 1 mM phenylmethylsulfonyl fluoride. Celldisruption was performed by three passages through a Microfluidizer(Microfluidics). Cell lysate was centrifuged twice in a GSA rotor to removecell debris, first at 10,000 rpm at 4 °C for 30 min, followed by 12,000 rpm at4 °C for 30 min. To collect the membrane pellet, the clarified supernatantwas ultracentrifuged in a type 45 Ti rotor at 43,000 rpm at 4 °C for 3 h. Themembrane fraction was resuspended in a buffer composed of 20 mM Hepes-NaOH (pH 8.0) and 300 mM NaCl, flash-frozen in liquid nitrogen, and sub-sequently stored at −80 °C.

All of the following steps were performed at 4 °C. Membrane proteinswere solubilized by addition of 2% (wt/vol) n-dodecyl-β-D-maltoside (β-DDM;Glycon Biochemicals) in 20 mM Hepes-NaOH (pH 8.0), 300 mM NaCl, and30 mM imidazole (pH 7.4) so that a membrane protein concentration of10 mg/mL was reached. After 2 h of rotation, the solubilized membraneproteins were separated by ultracentrifugation in a type 70.1 Ti rotorat 55,000 rpm for 2 h and filtered through a 0.22-μm centrifugal filter(Millipore). PfMATE was purified applying two chromatography steps. For affinitychromatography, the resulting supernatant containing the C-terminally His-tagged PfMATE was loaded onto a HisTrap HP column previously equili-brated with 20 mM Hepes-NaOH (pH 8.0), 300 mM NaCl, 30 mM imidazole(pH 7.4), and 0.05% (wt/vol) β-DDM. Protein peak fractions were eluted witha linear gradient of imidazole in purification buffer and concentrated usinga centrifugal filter device [30,000 molecular weight cutoff (MWCO)]. Theeluted protein sample was further purified by size exclusion chromatogra-phy at room temperature and loaded onto a Superdex 200 10/300 GL col-umn, which was previously washed with gel filtration buffer containing20 mM Hepes-NaOH (pH 8.0), 15 mM NaCl, and 0.06% (wt/vol) 6-cyclohexyl-1-hexyl-β-D-maltoside (Cymal-6; Anatrace). The protein peak fractions werepooled and concentrated with a centrifugal filter device (30,000 MWCO) to afinal concentration of 10–20 mg/mL for crystallization experiments.Outward-facing state (PDB ID codes 4MLB and 6HFB). The vectors containingtarget genes were transformed into E. coli TOP10 cells (Invitrogen), alongwith the pRARE plasmid (Novagen). The production and purification pro-cedures were essentially similar to the protocol described above with oneminor modification, namely, the size exclusion chromatography buffercontained 20 mM Hepes-NaOH (pH 8.0), 150 mM NaCl, and 0.05%pentaethylene glycol monodecyl ether (C10E5). Selenomethionine derivativeswere produced in modified E. coli LE392 cells in M9 media [43 mM Na2HPO4,23 mM KH2PO4, 8.5 mM NaCl, 19 mM NH4Cl, 1 mM MgCl2, 0.4% (wt/vol)glycerol, 30 mg/mL thiamine]. At first, the cells were cultured in M9 mediacontaining 2% casamino acids at 37 °C until the OD600 nm reached 1.0. Thesecells were harvested and washed twice with the same media. Later, thesewashed cells were inoculated in fresh M9 media containing 50 mg/mL L-selenomethionine, 50 mg/mL other amino acids, and 0.2% (wt/vol) arabinoseat 30 °C for 3 h to produce selenomethionine-labeled proteins. The purifi-cation procedure was similar, but 5 mM dithiothreitol (DTT) was added tothe purification buffer to avoid oxidation of selenium.

Crystallization.Outward-facing state (PDB ID codes 4MLB and 6HFB). Approximately 3,000 con-ditions were screened to crystallize wild-type PfMATE protein (PDB ID: 4MLB),and about 500 conditions were screened for each of the 17 variants (V62C,L69C, N95C, V109C, L144C, M170C, S177C, D184C, S235C, T318C, I324C,A411C, V435C, I438C, M446C, M170C/T318C, and N95C/M170C/S235C/T318C).As PfMATE contains no cysteines, these variants were prepared to help withphasing. All proteins were crystallized at 291 K by the sitting-drop vapordiffusion method. Crystallization droplets contain 10 mg/mL proteins in20 mM Hepes-NaOH (pH 8.0), 150 mM NaCl, 0.05% C10E5, 10 mM ytterbiumchloride, and 0.15% octyl-β-D-selenoglucoside, whereas the reservoir solu-tions were composed of 22–33% (wt/vol) polyethylene glycol 2,000 mono-methyl ether, and 0.1 M buffer solutions [N-(2-acetamido)iminodiacetic acid(ADA)·HCl or 2-(N-morpholino)ethanesulfonic acid (MES)·NaOH, pH 6.0–6.5].Crystals were obtained within 20–25 d. The selenomethionine-derivativeproteins were also crystallized using the same conditions in an anaerobicchamber in the presence of 5 mM DTT to avoid oxidation of selenium. Theselenomethionine-derivative crystals were always smaller and thinner than thewild-type crystals. Seven (V109C, V435C, T318C, M170C/T318C, E51C, S125C,and M126C) of 17 cysteine mutants were also crystallized in the same condi-tion. Heavy atom derivatives were prepared by cocrystallization and thesoaking method using HgCl2, CH3HgCl, C2H5HgCl, C6H5HgCl, CH3C6H4HgCl,

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(C2H5HgO)2HPO4, 4-(chloromercuri) benzenesulfonic acid (PCMBS), methylmercuric acetate, 4-(chloromercuri) benzoic acid, ethylmercury thiosalicylic acid(thimerosal), mersalyl acid, phenyl mercury acetate, tetrakis (acetoxymercuri)methane, K2HgI4, K2PtCl4, K2PtBr4, (NH4)2PtCl4, CH3PtI, platinum(II) tertpyr-idine chloride, (CH3)3PtI, platinum(IV) dichlorobis(1,2-ethanediamine-N,N′)dichloride, KAu(CN)2, aurothioglucose, trimethyllead acetate, triphenylleadacetate, hexaphenyldilead, lead acetate, OsCl3, K2OsO4, SmCl3, YbCl3, YCl3,BaCl2, NiCl2, ZnCl2, SrCl2, CdCl, CsCl, RbCl, NaBr, uranyl acetate, 5-amino-2,4,6-triiodoisophthalic acid, and xenon gas.

Crystals in the outward-facing conformation (6HFB) were generated bythe sitting-drop vapor diffusion method at 291 K. Crystallization dropletscontained 10 mg/mL protein in 20 mMHepes-NaOH (pH 8.0), 150 mMNaCl,0.05% (wt/vol) C10E5, and 0.15% (wt/vol) octyl-β-D-selenoglucoside (GlyconBiochemicals), whereas the reservoir solutions were composed of 30%(wt/vol) polyethylene glycol 2000 (PEG2000) monomethyl ether and0.1 M ADA·HCl (pH 6.5). Crystals matured to their full size within 20–25 d.The heavy atom derivative was prepared by soaking the crystals in1 M CsCl.Inward- and outward-facing states (PDB ID codes 6FHZ and 6GWH). The crystals ofPfMATE in the inward- and outward-facing conformations (6FHZ and 6GWH,respectively) were obtained by the LCP technique (30, 31). The protein so-lution in 20 mM Hepes-NaOH (pH 8.0), 15 mM NaCl, and 0.06% (wt/vol)Cymal-6 was filtered through a 0.22-μm spin filter (Millipore) before beingmixed with molten monoolein (9.9 MAG; Nu-Chek Prep) with a 2:3 (vol/vol)protein/monoolein ratio using a coupled syringe mixer. The 96-well crystal-lization trays were set up using a ProCrys Meso lipidic mesophase dispenser(Zinsser Analytic). The sizes of the precipitant and protein/monoolein solu-tion droplets were 1.5 μL and 100 nL, respectively. The use of a MemMesoHT-96 screen (Molecular Dimensions) resulted in successful crystallizationhits. The plates were stored at 295 K in the incubator of a CrystalMation(Rigaku) system.

For the inward-facing structure (6FHZ), the protein sample was incubatedwith the total lipid extract from P. furiosus after the affinity chromatographystep, subsequently copurified on a Superdex 200 10/300 GL column, and thencrystallized. Crystals of PfMATE in the inward-facing conformation weregrown in 0.1 M sodium chloride, 0.1 M magnesium chloride, 30% (vol/vol)PEG600, and 0.1 M sodium citrate (pH 5.0). PfMATE in the outward-facingstate was captured under almost identical conditions [0.1 M sodium chloride,30% (vol/vol) PEG500 DME, 0.1 M sodium citrate (pH 5.0)], however, in theabsence of the native lipids from P. furiosus. After crystals matured to theirfull size, they were harvested and directly flash-frozen in liquid nitrogenwithout an additional cryoprotectant.

Data Collection, Structure Determination, and Refinement.Outward-facing state (PDB ID codes 4MLB and 6HFB). In total, 140 diffractiondatasets were collected at beamlines BM14U, BM16, ID14-1, ID14-2, ID14-4,and ID23-1 of the European Synchrotron Radiation Facility (ESRF) andPXII/X10SA of the Swiss Light Source (SLS).

Two different crystal forms were obtained. Form-1 crystals belong to themonoclinic space group C2, and form-2 crystals belong to orthorhombicspace group I222. The 2.35-Å resolution native dataset was collected using aform-1 crystal at the BM16 beamline at the ESRF, which was obtained from10 mg/mL protein in a precipitant solution containing 24% (wt/vol)PEG2000 monomethyl ether, 0.1 M ADA·HCl (pH 6.5), and 10 mM YbCl3 as anadditive. A 5.9-Å resolution dataset was collected from a form-2 crystal atPXII/X10SA of the SLS, which was obtained from 10 mg/mL M170C/T318Cvariant in the precipitant solution containing 30% PEG2000 monomethylether, 0.1 M MES·NaOH (pH 6.0), 10 mM YbCl3, and 5 mM PCMBS. Back-soaking was not applied to this crystal because crystals were sometimesdamaged by this process.

All X-ray diffraction images were processed and scaled using XDS (32) andScala (33), respectively. For phasing purposes, form-1 native protein crystalsand form-2 M170C/T318C protein crystals were cocrystallized or soaked witha mercury compound mixture.

No reasonable solution was obtained by the molecular replacementmethod on the datasets of form-1 and form-2 crystals. Phasing by the SADmethod on the dataset of form-2 crystals was carried out by AutoSol softwarefrom the PHENIX program suite (34, 35). Eleven mercury atom sites wererefined, and initial phases and maps were calculated. Density modificationby RESOLVE (36) produced an electron density map at a resolution of 5.9 Å,in which the TMs and the outer membrane helices of the two PfMATEprotein molecules were clearly visible. Automatic modeling from this map byAutoBuild from PHENIX was not successful. The homology model built bythe web service Protein Homology/analogy Recognition Engine V 2.0 (Phyre2; http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (37) based on

the NorM_VC coordinates (3MKT) was manually fitted into the low-resolution map using Coot (38). The pseudosymmetrical N- and C-terminaldomains were distinguished using the electron density peaks of mercuryatoms at the introduced cysteine residues Cys170 and Cys318. This manuallymodified homology model was refined by phenix.refine. This refined modelwas used for molecular replacement utilizing the dataset of the form-1 crystal of the wild-type protein by Phaser-MR from PHENIX. Several solu-tions were obtained by the molecular replacement method using the Auto-Build program from PHENIX. One of the molecular replacement solutionsprovided an improved electron density map at a resolution of 2.5 Å, whichgave a good initial model at the automatic model building stage. The modelbuilt by AutoBuild was corrected manually using Coot. Structure refinementwas carried out using phenix.refine from PHENIX and Refmac (39–45) from theCCP4 suite (46). Finally, a 2.35-Å resolution structure was obtained.

In the case of the PfMATE outward-facing structure (6HFB), 4MLBwas usedfor the molecular replacement with Phaser (47–49) from the CCP4 suite. Themodel was manually corrected using Coot, and the structure refinement at aresolution limit of 3.5 Å was carried out using phenix.refine. The final modelhas 97.55% of the residues in the favored region in the Ramachandran plotand 2.34% of the residues in the allowed region.Inward- and outward-facing states (PDB ID codes 6FHZ and 6GWH). The X-ray dif-fraction datasets for PfMATE in the inward- and outward-facing conformations(6FHZ and 6GWH, respectively) were collected at the PXII/X10SA beamline ofthe synchrotron SLS. Data acquisition was performed at 100 K. The diffractiondata were processed with the XDS package for indexing, merging, and scaling.

For the determination of the outward-facing structure (6GWH), the PfMATEstructure (4MLB), namely, a monomer from the tetrameric asymmetrical unit,was used as the search model for the molecular replacement with Phaser. Thequality of the electron density map allowed a certain allocation of most of theamino acid residues, with the exception of the loop regions connectingTM3 and TM4 as well as TM9 and TM10. The model was subjected to manualadjustment using Coot, and subsequent iterative refinement at a resolutionlimit of 2.8 Å using phenix.refine. Parameters applied for the refinementstrategy included X/Y/Z coordinates, group B factors, occupancies with opti-mized X-ray/stereochemistry, and atomic displacement parameters weight. Inthe final model, 98.39% of the residues were in the favored region and therest were in the allowed region in the Ramachandran plot.

For the inward-facing structure determination (6FHZ), the initial phaseswere also obtained by molecular replacement. We split the same referencemodel (4MLB) at the center into two halves: the N-terminal fragment and theC-terminal fragment (TM1–TM6 and TM7–TM12, respectively) and usedthese two separate ensembles for the model generation in Phaser. Exceptfor the perturbed fragment of TM1, all TMs were assigned unambiguously.Due to the weak electron density map in the intramembrane region of TM1,the main chain tracing was validated by simulated annealing composite omitmaps and a feature-enhanced map generated by PHENIX (50–52). Aftermultiple cycles of manual model rebuilding in Coot, iterative refinement at aresolution limit of 2.8 Å was performed with phenix.refine. The final modelhas 97.03% of the residues in the favored region in the Ramachandran plotand 2.51% of the residues in the allowed region.

The quality of all models was assessed using MolProbity (53) andrefinement statistics.

The RMSD was calculated by TM-align (54). All structural figures wereprepared with PyMOL (55) and UCSF Chimera (56).

Cultivation of P. furiosus Cells. P. furiosus strain DSM3638 was grown underanaerobic conditions at 95 °C in 1/2 SME (Synthetisches Meerwasser/synthetic seawater) medium as described previously (57). Cultivation was per-formed in serum bottles as well as a 16-L bioreactor at the University ofRegensburg. Growth of P. furiosus was controlled by analyzing cell numbersusing a Thoma counting chamber with a 0.02-mm depth (Marienfeld). Cellswere harvested in the late exponential growth phase, flash-frozen in liquidnitrogen, and subsequently stored at −80 °C.

Determination of Archaeal Lipids by Mass Spectrometry. Lipids of P. furiosuswere extracted from the cells according to a methyl tert-butyl ether (MTBE)protocol (58). The lipid extract was injected on a Waters BEH C8 100 × 1-mm1.7-μm high performance liquid chromatography (HPLC) column used withan Ultimate 3000 UHPLC system (Thermo Fisher Scientific). Solvent A waswater with 1% ammonium acetate and 0.1% formic acid, and solvent Bwas acetonitrile/2-propanol 5:2 with 1% ammonium acetate and 0.1%formic acid. Gradient elution started at 50% mobile phase B, rising to100% B over 40 min; 100% B was held for 10 min, and the column wasreequilibrated with 50% B for 8 min before the next injection. The flowrate was 150 μL·min−1.

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