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Research Article

Inward-facing conformation of a multidrug resistance MATE family transporter

Sandra Zakrzewska, Ahmad Reza Mehdipour, Viveka Nand Malviya, Tsuyoshi Nonaka, Juergen Koepke, Cornelia Muenke, Winfried Hausner, View ORCID ProfileGerhard Hummer, Schara Safarian, and Hartmut Michel
  1. aDepartment of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
  2. bDepartment of Theoretical Biophysics, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
  3. cFaculty of Microbiology, University of Regensburg, 93053 Regensburg, Germany;
  4. dInstitute of Biophysics, Goethe University, 60438 Frankfurt am Main, Germany

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PNAS June 18, 2019 116 (25) 12275-12284; first published June 3, 2019; https://doi.org/10.1073/pnas.1904210116
Sandra Zakrzewska
aDepartment of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
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Ahmad Reza Mehdipour
bDepartment of Theoretical Biophysics, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
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Viveka Nand Malviya
aDepartment of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
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Tsuyoshi Nonaka
aDepartment of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
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Juergen Koepke
aDepartment of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
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Cornelia Muenke
aDepartment of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
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Winfried Hausner
cFaculty of Microbiology, University of Regensburg, 93053 Regensburg, Germany;
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Gerhard Hummer
bDepartment of Theoretical Biophysics, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
dInstitute of Biophysics, Goethe University, 60438 Frankfurt am Main, Germany
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  • ORCID record for Gerhard Hummer
Schara Safarian
aDepartment of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
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  • For correspondence: schara.safarian@biophys.mpg.de hartmut.michel@biophys.mpg.de
Hartmut Michel
aDepartment of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
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  • For correspondence: schara.safarian@biophys.mpg.de hartmut.michel@biophys.mpg.de
  1. Contributed by Hartmut Michel, May 2, 2019 (sent for review March 12, 2019; reviewed by Raimund Dutzler and José D. Faraldo-Gómez)

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Significance

Active efflux of drugs and toxic compounds carried out by multidrug and toxic compound extrusion (MATE) family transporters is one of the strategies developed by bacterial pathogens to confer multidrug resistance. To elucidate the underlying steps of the MATE transport cycle, structures of distinct intermediates are required; however, only structures in outward-facing conformations have been available. Our approach of using native lipids allowed trapping and visualization of the MATE transporter from Pyrococcus furiosus in an inward-facing conformation. This work highlights the importance of native lipids and opens an alternative view on the function and mechanism of action for the MATE family transporters.

Abstract

Multidrug and toxic compound extrusion (MATE) transporters mediate excretion of xenobiotics and toxic metabolites, thereby conferring multidrug resistance in bacterial pathogens and cancer cells. Structural information on the alternate conformational states and knowledge of the detailed mechanism of MATE transport are of great importance for drug development. However, the structures of MATE transporters are only known in V-shaped outward-facing conformations. Here, we present the crystal structure of a MATE transporter from Pyrococcus furiosus (PfMATE) in the long-sought-after inward-facing state, which was obtained after crystallization in the presence of native lipids. Transition from the outward-facing state to the inward-facing state involves rigid body movements of transmembrane helices (TMs) 2–6 and 8–12 to form an inverted V, facilitated by a loose binding of TM1 and TM7 to their respective bundles and their conformational flexibility. The inward-facing structure of PfMATE in combination with the outward-facing one supports an 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 distantly related families: multidrug and toxic compound extrusion (MATE), polysaccharide transporter (PST), oligosaccharidyl-lipid flippase (OLF), and mouse virulence factor (MVF) (1). Among these, the MATE transporters are most ubiquitous, being present in all domains of life (2). As secondary active transporters, they utilize transmembrane electrochemical ion gradients (Na+ or H+) (3⇓⇓–6) to drive the export of xenobiotics or cytotoxic metabolic waste products with specificity mainly for organic cations. MATE transporters confer resistance, for example, to fluoroquinolones, aminoglycoside antibiotics, and anticancer chemotherapeutical agents (4, 6), thus serving as promising drug targets in the fight against multidrug resistance. Based on their amino acid sequence similarity, the MATE family members are classified into the NorM, the DNA damage-inducible protein F (DinF), and the eukaryotic subfamilies (2). In recent years, the crystal structures of representative members of all three subfamilies have been published (7⇓⇓⇓⇓⇓⇓⇓–15). Since all of them represent only an outward-facing state, a detailed understanding of the complete transport cycle has remained elusive.

This work is focused on the DinF subfamily member, PfMATE (UniProtKB entry: Q8U2X0), which is one of the four annotated MATE transporters of the hyperthermophilic archaeon Pyrococcus furiosus. We could produce this protein in recombinant form in Escherichia coli, crystallize it, and determine the structure in an outward-facing conformation at the high resolution of 2.35 Å [Protein Data Bank (PDB) ID: 4MLB] (SI Appendix, Fig. S1). The structure of PfMATE is similar to NorM_VC from Vibrio cholerae and NorM_NG from Neisseria gonorrhoeae (7, 10), except for the electrostatic character of the substrate-binding cavity. TM1–TM6 and TM7–TM12 form helical bundles, called the N-lobe and C-lobe, respectively, creating a V-shaped cleft open to the extracellular side. However, opposite to the classical MATE transporters, the large central cavity of PfMATE is strongly positively charged (SI Appendix, Fig. S2A), which may be suited for accommodation of negatively charged lipophilic compounds, particularly lipids. Considering deviations between archaeal and bacterial lipids, we conducted crystallization experiments in the presence of native lipids extracted from P. furiosus. This approach led to the crystallization and structure determination of PfMATE in an inward-facing conformation, hinting at a crucial role of endogenous lipids in the functionality of this transporter. A loose binding of TM1 and TM7 to their respective lobes and their conformational flexibility appear to be key elements for the transition 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 native lipids, we also performed molecular dynamics (MD) simulations in an archaeal-type lipid bilayer. Furthermore, the combination of our data from single-wavelength anomalous dispersion (SAD) experiments using anomalous signals of Cs+ with the results of MD simulations indicates the presence of a monovalent cation-binding site at Asp41. Based on these data, we could establish a framework of the structural changes that occur during the transition between the inward- and outward-facing conformations of the MATE transporter.

Results

Crystal Structure of PfMATE in the Inward-Facing Conformation.

Our PfMATE structure in an outward-facing state (PDB ID: 4MLB) revealed that the V-shaped central vestibule contains four positively charged residues (Arg83, Arg161, Arg284, and Arg402). Furthermore, the interior of the central cavity is lined with a cluster of methionine residues (Met27, Met31, Met64, Met173, Met256, Met260, Met287, and Met381), creating a hydrophobic environment around the center of the membrane. Remarkably, the positively charged cavity of PfMATE shows a reverse polarity compared with the canonical MATE transporters, such as NorM_NG and NorM_VC, as well as the eukaryotic MATEs. Among the MOP superfamily, the PfMATE vestibule rather resembles 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 not from E. coli, led to structure determination of PfMATE in an inward-facing conformation with the central cavity accessible for substrates from the intracellular side (Fig. 1A and SI Appendix, Table S2). The prominent feature of this conformation is the significantly bent TM1, which undergoes a notable secondary structure rearrangement (SI Appendix, Fig. S3). A partial unwinding of this helix hints at a high degree of flexibility, suggesting a role in the conformational switch between outward- and inward-oriented states. A structural superposition between the N-lobe and C-lobe of the inward-facing structure [global Cα root-mean-square deviation (RMSD) = 2.58 Å] demonstrates that the internal twofold symmetry between these two domains remains preserved (Fig. 1B). The conformational rearrangement of TM7 of the C-lobe is similar to that of its symmetry-related counterpart TM1 of the N-lobe, which undergoes substantial bending and is located in close proximity to the C-lobe helices (Fig. 1B). These structural alterations cause the intracellular opening to be significantly wider (about 26 Å) than the extracellular one (about 18 Å) in the respective access states (Fig. 1C).

Fig. 1.
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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 represent the 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 the N-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 of the IFC shows their symmetrical arrangement. (C) Surface representation of the OFC and IFC showing the central binding cavity from the top and in cross-section.

Structural Basis of the Extracellular and Intracellular Barrier Formation.

Fundamentally, secondary active transporters use electrochemical gradients of either protons or sodium ions to enable the uphill movement of substrates across the membrane. According to the basic principles of the alternating access model (17, 18), MATE transporters sequentially expose their substrate-binding cavity to either side of the membrane during a transport cycle by internal or external barrier formation. These barriers restrict the accessibility of the substrate- and ion-binding sites to one respective surrounding at a time. A structural alignment of the N-lobes (Cα RMSD = 2.02 Å) and C-lobes (Cα RMSD = 1.84 Å) of the PfMATE outward- and inward-facing conformations reveals that these barriers are formed by local and global conformational changes, including rigid body movements of the N- and C-lobes, a hinge-like motion of the N-terminal segment of TM1, and unwinding of TM1 halfway across the membrane. Alternating access to the substrate-binding site appears to be enabled by significant movements of TM1 and TM7, while the remaining TMs of the N-lobe (TM 2–TM6) and the C-lobe (TM8–TM12) stay together and undergo a rigid body movement.

In the inward-facing conformation, the extracellular barrier is mediated predominantly by hydrophobic and aromatic interactions between residues located at the extracellular apex of the lobes. This tightly closed barrier with a thickness of about 10 Å involves hydrophobic interactions between Pro50 (TM2), Leu53 (TM2), Ala54 (TM2), Ala277 (TM8), Ala355 (loop connecting TM9 and TM10), Val357 (TM10), and Ile358 (TM10) (SI Appendix, Fig. S4A). The van der Waals interactions between Phe349 (TM9) and Ile43 (TM1), Gly49 (TM1), and Ile285 (TM8) serve as key components for the formation of the extracellular barrier, which blocks access to the substrate-binding site from the extracellular side of the membrane. The displacement of TM7 and TM8 at the extracellular apex of the C-lobe from their positions in the outward-facing structure, closely approaching the helices of the N-lobe, is probably due to the hydrophobic interactions between 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 extracellular gate and the formation of a hydrophobic groove within the C-lobe. A network of interactions between several residues from the C-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 hydrogen bond between the carbonyl oxygen atom of Ile43 (TM1), the side-chain OH group of Tyr351 (loop between TM9 and TM10), and the 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 the closure of the extracellular barrier has to be disrupted. Formation of the intracellular barrier obstructs solvent accessibility from the cytoplasm, while the extracellular barrier opens (Movie S1). Thus, the substrate- and ion-binding sites are accessible via an aqueous cavity, which is exposed to the extracellular side of the membrane. The thicker barrier between the substrate and the cytoplasm (about 14 Å) (Fig. 2A) may be correlated with the substantial movement of the intracellular half of TM1. Consistent with the positive inside rule (19), the intracellular side of the PfMATE contains more positively charged residues compared with the extracellular barrier. Therefore, not only hydrophobic interactions, which are predominant in the extracellular barrier, but also ionic interactions, such as the salt bridge formed between the side chains of Arg244 (TM7) and Glu393 (TM11), strengthen the intracellular barrier. The ionic interactions between the side chains of Glu310 (TM8), Arg13 (TM1), and Arg88 (TM2) are likely to be the driving factor for the movement of TM8 and TM9 on the intracellular side, thereby closing the intracellular pathway (SI Appendix, Fig. S4C). In the outward-facing structure, the side chain of Arg244 (TM7) is flipped away from Asp241 (TM7) and forms an ionic interaction with the side chain 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 intracellular barrier. This coordination of two positively charged residues may imply the existence of a repulsive electrostatic gate, which could preclude a flux of protons and other cations, thereby forming a tight barrier preventing ion leakage during the conformational transition. The hydrophobic interaction between Val9 (TM1) and Ile85 (TM2) is another major contributor in the formation of the intracellular barrier, which stabilizes the position of the short helical stretch of the N terminus in the outward-facing structure. An aqueous path to the intracellular side is also closed through a hydrophobic interaction network between Ala82, Gly86 (TM2), and Ala306 (TM8). The outward- and inward-facing conformations of PfMATE illustrate how these two distinct barriers are formed by a network of mostly hydrophobic residues at the closely interacting lobe interfaces in each state.

Fig. 2.
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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-facing and outward-facing conformations. Water access to the cavity is restricted to the inside and outside, respectively. (B) Distance between the sodium ion-binding site (carboxyl oxygen atoms of Asp41) and a Na+ that binds spontaneously during MD simulations of the outward-facing conformation (OFC) state (Top) and that remained 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. The purple 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 the crystal 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 coordinating residues are shown in stick representation, whereas the blue mesh shows the anomalous peak for Cs+.

Structural and Functional Implications of TM1 Bending.

In the previously proposed model of the PfMATE transport, the highly conserved Asp41 constitutes the key residue associated with proton coupling (14). It has been proposed that Asp41 is deprotonated at high pH and that its protonation induces the conformational change of TM1 from the straight state to the bent state. This conclusion was based on two PfMATE crystal structures obtained at high pH (7.0–8.0) and low pH (6.0–6.5), respectively. The aforementioned interpretation raised some criticism, leading to the hypothesis about TM1 bending to be rather affected by interactions with exogenous lipids (monoolein) present in the crystallization conditions as described previously (13, 20). This discrepancy motivated us to evaluate the relationship between pH and structural alteration of TM1. Therefore, we determined outward-facing structures of PfMATE from crystals generated at low pH (5.0 and 6.5) by both vapor diffusion and lipidic cubic phase (LCP) methods (SI Appendix, Fig. S7). A comparison between these structures and the previously presented high pH outward-facing structure (PDB ID: 3VVN) reveals that TM1 remains straight irrespective of the pH of the crystallization media; there are no major structural differences, as reflected by a very low Cα RMSD = 0.86 Å of the 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 chains A, 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, respectively, (PDB ID: 6HFB) of the structures obtained in the absence of monoolein molecules. Hence, our results are consistent with the notion that the conformational rearrangements of helix TM1 are most likely independent of pH. In recently published MD simulations of PfMATE with bent TM1 (PDB ID: 3VVO) in the outward-facing conformation, replacing a water molecule near Asp41 by sodium produced a stable structure (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 at Gly30 allows the large hinge-like movement within the N-terminal segment of TM1 to adopt the inward-facing conformation. The unfolded intramembrane region in TM1 exhibits remarkable flexibility, suitable for coordination of substrates or ions. During the transition from the outward- to inward-facing state, two segments of TM1, one from Ala17 to Ala31 and the second from Ser32 to Val45, tilt relative to each other. This tilting can be decomposed into two movements described by the following angles: The first angle defines the movement of TM1 as the gatekeeper of the lateral opening in the Y/Z plane (about 27°), while the second angle describes the pronounced movement in the X/Z plane (about 42°), showing that the intramembrane half of TM1 also acts as a plug to seal the lateral opening in the outward-facing conformation. In the inward-facing structure, the side chain of Phe60 (TM2) is flipped toward Val56 (TM2), also creating a hydrophobic contact with Gly42 (TM1), which leads to a helical kink. In the outward-facing structure, these residues are shifted apart and TM1 remains straight. The bending of TM1 in the inward-facing state is further facilitated by side-chain interactions between Asn154 (TM10) and Gln34 (TM1). A hydrogen bond between the side-chain carboxyl group of Asp41 (TM1) and the side-chain hydroxyl group of Tyr139 (TM4), as well as a hydrophobic interaction between Pro26 (TM1) and Ala166 (TM5), is most likely involved in the formation of the kinked state of TM1. The substantial movements of TM1 toward the C-lobe in the inward-facing state appear to contribute also to the widened intracellular opening compared with the extracellular state (Fig. 1C). The structural alteration of TM1 in the inward-facing structure 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 transporters harnessing the energy from an electrochemical transmembrane ion gradient (Na+ or H+) to enable substrate transport. The ion-binding site in the outward-facing conformation of PfMATE is located near Asp41, Asn180, Asp184, and Thr202 close to the surface of the internal cavity aqueous path leading to the ion-binding site in the outward-facing structure, and it is placed in a water-accessible surrounding, whereas in the inward-facing conformation, it appears to be occluded. Sodium ions and water molecules are not straightforwardly distinguishable through X-ray crystallography. Tanaka et al. (14) interpreted an electron density peak near this site as a bound water molecule in the outward-facing state (PDB ID: 3VVO and 3VVN). A highly similar ion-binding site formed by Asp35, Asn174, Asp178, Ala192, and Thr196 (corresponding to the homologous residues Asp41, Asn180, Asp184, Ala198, and Thr202 of PfMATE) was identified in the X-ray structure of another MATE transporter from V. cholerae, VcmN (8). The electron density signal at this site was also assigned to a water molecule by analogy with the aforementioned studies (14). Our CsCl heavy atom derivative dataset of the outward-facing structure (PDB ID: 6HFB) shows an anomalous difference electron density for a Cs+, adjacent to the carboxyl oxygen atom Oδ1 of Asp41 (Fig. 2E). Presumably, due to the larger size, cesium ions do not occupy exactly the same position as a Na+. The anomalous signal from a Cs+ demonstrates the negative electrostatic potential of this site, which is suited for binding monovalent cations. As shown in the recent MD simulations, the outward-facing bent state of PfMATE (PDB ID: 3VVO) reveals a highly coordinated Na+-binding site (21). This sodium ion-occluded bent state appears to be an intermediate in the transport cycle stabilized by ion binding before transition into the inward-facing state. Our MD simulations of the outward-facing structure showed spontaneous Na+ binding to the Cs+ site of the crystal structure (Fig. 2B). After ion recognition, the binding site did not immediately close and the sodium ion formed four interactions with the protein (two with Asp41 and one each with Asn180 and Thr202), but retained two water molecules. Later during 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 state and 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 fully coordinated state, TM1 is slightly bent, adopting an intermediate conformation between those of 3VVO and 3VVN.

A prominent spherical electron density signal was also observed in the Fo-Fc map of the inward-facing structure in very close proximity to Asp41 (Fig. 2C), which is consistent with a coordination number of six. This observation raises the possibility that the electron density in the center is caused by the presence of a Na+ ion. Also, the coordination is highly similar to that of Na+ sites in other proteins of known structure (22, 23). The distances between the sodium ion and oxygen atoms of the coordinating residues in this structure [Oδ1 of Asp41 (2.42 Å), Oδ1 of Asn180 (2.45 Å), Oδ1 of Asp184 (2.60 Å), carbonyl oxygen atom of Ala198 (2.08 Å), and Oγ of Thr202 (2.38 Å)] also indicate a Na+-bound state. The presence of a monovalent cation-binding site in the N-lobe of PfMATE was supported by our MD simulations, which showed a Na+-binding site near Asp41 (pKa = 5.1) and Asp184 (pKa > 14) in the outward-facing structure. The calculated pKa values for the inward-facing structure strongly imply that in the absence of sodium ions, both Asp41 (pKa = 13.7) and Asp184 (pKa > 14) are protonated, while in the presence of sodium ions, Asp41 (pKa = 0) is most likely charged (Fig. 2D). Results obtained from the MD simulations in the presence of sodium ions indicate a well-coordinated binding site, while in the absence of sodium ions, the protonation of both residues, Asp41 and Asp184, is essential for the stability of the binding site (Fig. 2 B and D). In light of the high calculated pKa of Asp41 in the inward-facing structure (pKa = 13.7 in the absence of sodium ions), Na+ release could be coupled to Asp41 protonation. Structuring of the unwound TM1 segment could be another relevant factor. The structural data and the results of the MD simulations imply a sodium ion-dependent transport mechanism of PfMATE. However, we cannot exclude a dual specificity for sodium ions and protons, which was proposed in the recent publications demonstrating a dual ion coupling behavior for NorM_VC (24, 25).

Surprisingly, during the MD runs, we also observed spontaneous binding of chloride ions into the cavity in the inward-facing structure (SI Appendix, Fig. S8). The chloride interacts with Arg284, Thr35, Asn38, Trp283, and Met287. Interestingly, a Cl− has also been observed near Arg24 and Arg255 in the MurJ inward-facing structure (16), which corresponds to a similar position of PfMATE after superposition of both structures. We also observed Cl− binding into the outward-facing structure in a different site near Arg402. In the archaeal lipid simulation, the lipid head group subsequently replaced the Cl− ion. By contrast, in the bacterial lipid simulation, after 1.2 μs, both a lipid and a chloride ion were bound. Currently, the functional importance of the chloride ion for PfMATE remains unclear. We cannot exclude that the bound chloride is released to the external side during the transition to the outward-facing state in such a way that MATE (or, in general, MOP) transporters are actually chloride/sodium ion antiporters. The cotransport of a chloride ion would be energetically beneficial by contributing driving force to the transport.

Interaction of Archaeal Lipids with PfMATE.

Lengthy electron densities in the map of the inward-facing conformation observed in proximity to the intramembrane region of TM1 and helices TM8 and TM9 may originate from the lipid molecules. Since, at the present resolution, these densities cannot be unambiguously assigned to lipids, they were not included during the structure refinement. It is commonly known that lipids are very often poorly resolved and/or difficult to distinguish from other molecules like detergents. The local environment for each lipid can influence the conformation of the lipid tails, their mobility, and disorder, leading to variable conformations. Due to their flexibility or multiple alternative conformations, lipids can result in weak signals in the electron density maps. Therefore, structural information on the lipid–protein interaction is difficult to obtain. We cannot empirically identify a specific lipid-binding site and figure out how exactly the addition of the native lipids leads to crystallization of this protein in the inward-facing state. Nevertheless, having established that native lipids act like conformational modulators of PfMATE, we carried out all-atom MD simulations of this protein embedded in a lipid bilayer 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 certain mechanisms, which allow surviving under extreme conditions. One of the fundamental thermal adaptations is the membrane lipid composition (27). Our mass spectrometric analysis reveals that P. furiosus membranes contain diphytanyl phosphatidyl inositol (DPI), diphytanyl phosphatidyl N-acetyl hexose, diphytanyl phosphatidyl glycerol (DPG), diphytanyl phosphatidic acid (DPA), and isoprenoidal glycerol dialkyl glycerol tetraether lipids. Based on these results, we performed MD simulations for the interaction studies of PfMATE and lipids under native-like conditions at an elevated temperature (100 °C) (Movie S2). PfMATE was embedded in an archaeal lipid bilayer, consisting of DPI, DPG, and DPA lipid species (ratio of 45:20:35%). Our MD simulation results for the outward-facing state show lipid molecules moving into the cavity and being accommodated by the positively charged pocket. Interestingly, during several different MD simulation runs, we observed a complete reorientation of negatively charged lipids (mostly PG species) inside the cavity, with the head group moiety pointing toward the bottom of the cavity (Fig. 3). In these simulations, the lipid entered the cavity from the outer leaflet. Initially, the lipid head group intruded into the cavity several times and then moved back and interacted mainly with Arg284 and Trp283; subsequently, a predominantly electrostatic interaction between the head group and Arg402 resulted in a further movement of the head group inside the cavity, which finally triggered the full reorientation of the 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 cluster of methionine residues, including Met31, Met64, Met256, Met260, and Met287, coordinated the aliphatic tails of the lipid. The lipid remained bound into the cavity for the rest of the simulation (up to 1.5 μs), with its headgroup stabilized by hydrogen bonds and electrostatic interactions and its tail stabilized by abundant hydrophobic interactions with the protein cavity. Lipid binding was observed only for the outward-facing structure. In the inward-facing structure, closed lateral gates block lipid access to the cavity. Interestingly, we observed the simultaneous lipid and sodium ion binding in the outward-facing state. Our results demonstrate the existence of a pathway for lipid access into the central cavity of PfMATE and the possibility of lipid flipping.

Fig. 3.
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Fig. 3.

Spontaneous binding and reorientation of an archaeal lipid in MD simulations of PfMATE. (Left) Side view of the representative MD snapshot showing 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 the outside leaflet at the top and spontaneously flipped its orientation. (Right) Zoom-in view highlights residues contacting the lipid. MD snapshots have been taken from the end of the 1.5-μs simulation.

Discussion

We present here the inward-facing structure of a MATE family transporter, namely, PfMATE, from P. furiosus. This structure has to be expected to possess a high-affinity substrate-binding state that is not only essential from the physiological perspective, but also may serve for the structure-aided design of inhibitors. Together with the MD simulations, our structural data imply lipid-specific regulation of conformational rearrangements during the alternating access mechanism of action of this transporter. Tanaka et al. (14) reported two outward-open structures of PfMATE and convincingly presented data that PfMATE confers norfloxacin resistance to the E. coli BW25113 ΔacrB strain. Interestingly, the lipid or monoolein molecules were observed in the straight and bent outward-facing structures of PfMATE (3VVO and 3VVN, respectively). The binding site of two monoolein molecules observed exclusively in the N-lobe of their straight structure (3VVN) corresponds to the drug recognition site in the Br-norfloxacin–bound structure (3VVP). The authors suggested that these molecules may mimic the hydrophobic substrates. Based on our functional studies, the minimum inhibitory concentration (MIC) values were determined for several antimicrobial compounds, including antibiotics and DNA-binding dyes in the E. coli KAM32 strain (ΔacrB, ΔydhE, and hsdΔ5) and three variants of the BW25113 strain, namely, BW25113A (ΔacrAB, ΔtolC, ΔydhE, ΔmdfA, and ΔemrE), BW25113B (ΔacrAB, ΔydhE, and ΔemrE), and BW25113C (ΔacrB) (SI Appendix, Fig. S5 and Table S1). The MIC values for different compounds were the same for the test cells containing the plasmid with the gene coding for PfMATE (pBAD-PfMATE) and the control cells containing the empty vector (pBAD). Overexpression of PfMATE did not provide any additional resistance against any of the tested compounds, implying that this transporter may be more specialized than the classical MATE family members. Importantly, only the E. coli C43(DE3) ΔacrAB strain containing pBAD-PfMATE showed some increased norfloxacin resistance compared with the cells with the empty vector; however, the difference is not as significant compared with the cells containing the gene coding for the MATE transporter NorM_VP from Vibrio parahaemolyticus used as a positive control (SI Appendix, Fig. S6). Our results are also similar to those of functional studies on ClbM transporter from the MATE family, which did not provide resistance to ethidium bromide in E. coli KAM32 cells; however, a resistance was observed in E. coli C43(DE3) ΔacrAB cells. Despite the high structural resemblance to the other MATE family transporters, particularly PfMATE, ClbM has an additional unique function of precolibactin transport (12). Therefore, we cannot exclude that the primary function of PfMATE is lipid transport. The interaction of lipid molecules with PfMATE also opens an alternative view on the mechanism of action for the MATE family members, in which specific lipid species could either modulate the transition between the outward- and inward-facing state or mimic the substrates for these transporters. It is noteworthy that Martens et al. (28) recently proposed a model for the lipid-induced conformational equilibrium of secondary active transporters based on hydrogen/deuterium exchange mass spectrometry and MD simulation experiments. This work particularly described how lipid binding to conserved networks of charged residues induces the conformational transition toward an inward-facing conformation. Our results comprise fundaments for future structural and functional studies 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 our structural and computational studies thus pave the way toward a better understanding of the underlying steps of the PfMATE transport cycle and strongly favor the conclusion that native lipids from the corresponding source organism and their specific interactions with protein may be of physiological relevance and essential for function and conformational regulation of MATE transporters in general.

Methods

Gene Cloning and Mutagenesis.

Genomic DNA of P. furiosus Vc 1 was obtained from Michael Thomm and Harald Huber, Faculty of Microbiology, University of Regensburg. The target gene PfMATE (UniProtKB entry Q8U2X0) was cloned 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 protein were 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 variants were also prepared (11ML/M170C/M446C, 12ML/M170C, 12ML/M446C, and 13ML), 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) were substituted by leucine residues and M170 and M446 were substituted by either cysteine or leucine residues. These constructs were prepared to obtain mercury and selenomethionine derivatives.

PfMATE Production and Purification.

Inward- and outward-facing states (PDB ID codes 6FHZ and 6GWH).

The gene encoding PfMATE was expressed in E. coli TOP10 cells (Invitrogen). The cultures were grown in lysogeny broth (LB) medium supplemented with 50 μg/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 induced by addition of l-arabinose to a final concentration of 0.02% (wt/vol). After 3 h of incubation at 37 °C, cells were harvested by centrifugation at 4 °C for 15 min. Bacterial cell pellets were resuspended in a fivefold excess (vol/wt) of lysis buffer composed of 20 mM Hepes-NaOH (pH 8.0), 300 mM NaCl, 5 mM MgCl2, a pinch of DNaseI, and 1 mM phenylmethylsulfonyl fluoride. Cell disruption was performed by three passages through a Microfluidizer (Microfluidics). Cell lysate was centrifuged twice in a GSA rotor to remove cell debris, first at 10,000 rpm at 4 °C for 30 min, followed by 12,000 rpm at 4 °C for 30 min. To collect the membrane pellet, the clarified supernatant was ultracentrifuged in a type 45 Ti rotor at 43,000 rpm at 4 °C for 3 h. The membrane 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 subsequently stored at −80 °C.

All of the following steps were performed at 4 °C. Membrane proteins were 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, and 30 mM imidazole (pH 7.4) so that a membrane protein concentration of 10 mg/mL was reached. After 2 h of rotation, the solubilized membrane proteins were separated by ultracentrifugation in a type 70.1 Ti rotor at 55,000 rpm for 2 h and filtered through a 0.22-μm centrifugal filter (Millipore). PfMATE was purified applying two chromatography steps. For affinity chromatography, the resulting supernatant containing the C-terminally His-tagged PfMATE was loaded onto a HisTrap HP column previously equilibrated 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 with a linear gradient of imidazole in purification buffer and concentrated using a centrifugal filter device [30,000 molecular weight cutoff (MWCO)]. The eluted protein sample was further purified by size exclusion chromatography at room temperature and loaded onto a Superdex 200 10/300 GL column, which was previously washed with gel filtration buffer containing 20 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 were pooled and concentrated with a centrifugal filter device (30,000 MWCO) to a final concentration of 10–20 mg/mL for crystallization experiments.

Outward-facing state (PDB ID codes 4MLB and 6HFB).

The vectors containing target genes were transformed into E. coli TOP10 cells (Invitrogen), along with the pRARE plasmid (Novagen). The production and purification procedures were essentially similar to the protocol described above with one minor modification, namely, the size exclusion chromatography buffer contained 20 mM Hepes-NaOH (pH 8.0), 150 mM NaCl, and 0.05% pentaethylene glycol monodecyl ether (C10E5). Selenomethionine derivatives were 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 media containing 2% casamino acids at 37 °C until the OD600 nm reached 1.0. These cells were harvested and washed twice with the same media. Later, these washed cells were inoculated in fresh M9 media containing 50 mg/mL l-selenomethionine, 50 mg/mL other amino acids, and 0.2% (wt/vol) arabinose at 30 °C for 3 h to produce selenomethionine-labeled proteins. The purification procedure was similar, but 5 mM dithiothreitol (DTT) was added to the purification buffer to avoid oxidation of selenium.

Crystallization.

Outward-facing state (PDB ID codes 4MLB and 6HFB).

Approximately 3,000 conditions 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 with phasing. All proteins were crystallized at 291 K by the sitting-drop vapor diffusion method. Crystallization droplets contain 10 mg/mL proteins in 20 mM Hepes-NaOH (pH 8.0), 150 mM NaCl, 0.05% C10E5, 10 mM ytterbium chloride, and 0.15% octyl-β-d-selenoglucoside, whereas the reservoir solutions were composed of 22–33% (wt/vol) polyethylene glycol 2,000 monomethyl 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-derivative proteins were also crystallized using the same conditions in an anaerobic chamber in the presence of 5 mM DTT to avoid oxidation of selenium. The selenomethionine-derivative crystals were always smaller and thinner than the wild-type crystals. Seven (V109C, V435C, T318C, M170C/T318C, E51C, S125C, and M126C) of 17 cysteine mutants were also crystallized in the same condition. Heavy atom derivatives were prepared by cocrystallization and the soaking method using HgCl2, CH3HgCl, C2H5HgCl, C6H5HgCl, CH3C6H4HgCl, (C2H5HgO)2HPO4, 4-(chloromercuri) benzenesulfonic acid (PCMBS), methyl mercuric 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) tertpyridine chloride, (CH3)3PtI, platinum(IV) dichlorobis(1,2-ethanediamine-N,N′) dichloride, KAu(CN)2, aurothioglucose, trimethyllead acetate, triphenyllead acetate, 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 by the sitting-drop vapor diffusion method at 291 K. Crystallization droplets contained 10 mg/mL protein in 20 mM Hepes-NaOH (pH 8.0), 150 mM NaCl, 0.05% (wt/vol) C10E5, and 0.15% (wt/vol) octyl-β-d-selenoglucoside (Glycon Biochemicals), whereas the reservoir solutions were composed of 30% (wt/vol) polyethylene glycol 2000 (PEG2000) monomethyl ether and 0.1 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 in 1 M CsCl.

Inward- and outward-facing states (PDB ID codes 6FHZ and 6GWH).

The crystals of PfMATE in the inward- and outward-facing conformations (6FHZ and 6GWH, respectively) were obtained by the LCP technique (30, 31). The protein solution 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 being mixed 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 crystallization trays were set up using a ProCrys Meso lipidic mesophase dispenser (Zinsser Analytic). The sizes of the precipitant and protein/monoolein solution droplets were 1.5 μL and 100 nL, respectively. The use of a MemMeso HT-96 screen (Molecular Dimensions) resulted in successful crystallization hits. 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 incubated with the total lipid extract from P. furiosus after the affinity chromatography step, subsequently copurified on a Superdex 200 10/300 GL column, and then crystallized. Crystals of PfMATE in the inward-facing conformation were grown 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-facing state 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 the absence of the native lipids from P. furiosus. After crystals matured to their full size, they were harvested and directly flash-frozen in liquid nitrogen without an additional cryoprotectant.

Data Collection, Structure Determination, and Refinement.

Outward-facing state (PDB ID codes 4MLB and 6HFB).

In total, 140 diffraction datasets were collected at beamlines BM14U, BM16, ID14-1, ID14-2, ID14-4, and ID23-1 of the European Synchrotron Radiation Facility (ESRF) and PXII/X10SA of the Swiss Light Source (SLS).

Two different crystal forms were obtained. Form-1 crystals belong to the monoclinic space group C2, and form-2 crystals belong to orthorhombic space group I222. The 2.35-Å resolution native dataset was collected using a form-1 crystal at the BM16 beamline at the ESRF, which was obtained from 10 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 an additive. A 5.9-Å resolution dataset was collected from a form-2 crystal at PXII/X10SA of the SLS, which was obtained from 10 mg/mL M170C/T318C variant in the precipitant solution containing 30% PEG2000 monomethyl ether, 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 sometimes damaged by this process.

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

No reasonable solution was obtained by the molecular replacement method on the datasets of form-1 and form-2 crystals. Phasing by the SAD method on the dataset of form-2 crystals was carried out by AutoSol software from the PHENIX program suite (34, 35). Eleven mercury atom sites were refined, and initial phases and maps were calculated. Density modification by 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 PfMATE protein molecules were clearly visible. Automatic modeling from this map by AutoBuild from PHENIX was not successful. The homology model built by the web service Protein Homology/analogy Recognition Engine V 2.0 (Phyre 2; 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-terminal domains were distinguished using the electron density peaks of mercury atoms at the introduced cysteine residues Cys170 and Cys318. This manually modified homology model was refined by phenix.refine. This refined model was used for molecular replacement utilizing the dataset of the form-1 crystal of the wild-type protein by Phaser-MR from PHENIX. Several solutions were obtained by the molecular replacement method using the AutoBuild program from PHENIX. One of the molecular replacement solutions provided an improved electron density map at a resolution of 2.5 Å, which gave a good initial model at the automatic model building stage. The model built by AutoBuild was corrected manually using Coot. Structure refinement was carried out using phenix.refine from PHENIX and Refmac (39⇓⇓⇓⇓⇓–45) from the CCP4 suite (46). Finally, a 2.35-Å resolution structure was obtained.

In the case of the PfMATE outward-facing structure (6HFB), 4MLB was used for the molecular replacement with Phaser (47⇓–49) from the CCP4 suite. The model was manually corrected using Coot, and the structure refinement at a resolution limit of 3.5 Å was carried out using phenix.refine. The final model has 97.55% of the residues in the favored region in the Ramachandran plot and 2.34% of the residues in the allowed region.

Inward- and outward-facing states (PDB ID codes 6FHZ and 6GWH).

The X-ray diffraction datasets for PfMATE in the inward- and outward-facing conformations (6FHZ and 6GWH, respectively) were collected at the PXII/X10SA beamline of the synchrotron SLS. Data acquisition was performed at 100 K. The diffraction data were processed with the XDS package for indexing, merging, and scaling.

For the determination of the outward-facing structure (6GWH), the PfMATE structure (4MLB), namely, a monomer from the tetrameric asymmetrical unit, was used as the search model for the molecular replacement with Phaser. The quality of the electron density map allowed a certain allocation of most of the amino acid residues, with the exception of the loop regions connecting TM3 and TM4 as well as TM9 and TM10. The model was subjected to manual adjustment using Coot, and subsequent iterative refinement at a resolution limit of 2.8 Å using phenix.refine. Parameters applied for the refinement strategy included X/Y/Z coordinates, group B factors, occupancies with optimized X-ray/stereochemistry, and atomic displacement parameters weight. In the final model, 98.39% of the residues were in the favored region and the rest were in the allowed region in the Ramachandran plot.

For the inward-facing structure determination (6FHZ), the initial phases were also obtained by molecular replacement. We split the same reference model (4MLB) at the center into two halves: the N-terminal fragment and the C-terminal fragment (TM1–TM6 and TM7–TM12, respectively) and used these two separate ensembles for the model generation in Phaser. Except for 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 omit maps and a feature-enhanced map generated by PHENIX (50⇓–52). After multiple cycles of manual model rebuilding in Coot, iterative refinement at a resolution limit of 2.8 Å was performed with phenix.refine. The final model has 97.03% of the residues in the favored region in the Ramachandran plot and 2.51% of the residues in the allowed region.

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

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

Cultivation of P. furiosus Cells.

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

Determination of Archaeal Lipids by Mass Spectrometry.

Lipids of P. furiosus were 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-mm 1.7-μm high performance liquid chromatography (HPLC) column used with an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). Solvent A was water with 1% ammonium acetate and 0.1% formic acid, and solvent B was acetonitrile/2-propanol 5:2 with 1% ammonium acetate and 0.1% formic acid. Gradient elution started at 50% mobile phase B, rising to 100% B over 40 min; 100% B was held for 10 min, and the column was reequilibrated with 50% B for 8 min before the next injection. The flow rate was 150 μL⋅min−1.

Data acquisition was performed according to a previously reported protocol (59) by means of an Orbitrap mass spectrometer (LTQ-Orbitrap; Thermo Fisher Scientific) full scan in preview mode at a resolution of 100,000 and <2 ppm mass accuracy with external calibration. The spray voltage was set to 4,500 V, and the capillary temperature was at 300 °C. From the Fourier transform mass spectrometry preview scan, the 10 most abundant m/z values were picked in data-dependent acquisition mode, fragmented in the linear ion trap analyzer, and ejected at nominal mass resolution. Normalized collision energy was set to 50%, the repeat count was 2, and the exclusion duration was set at 10 s. Data analysis was performed using Lipid Data Analyzer, a custom-developed software tool (60, 61).

MD Simulations.

The two PfMATE structures were embedded in lipid bilayers of varying composition using CHARMM-GUI (62). Based on multiconformation continuum electrostatics (MCCE) (63, 64) calculations at pH 7, the protonation states of the titrable Asp, Glu, and His residues were assigned. Asp184 and Glu331 were assigned as neutral in the outward-facing conformation, while the other Asp and Glu residues were assigned as charged. In the inward-facing conformation, Asp41, Asp184, Glu163, Glu273, and Glu331 were protonated in the absence of a sodium ion near Asp41/Asp184, whereas in the presence of a sodium ion, Asp41 was charged. All His residues were neutral, and they were protonated at their either the Nδ or Nε atom according to the network of hydrogen bonds.

All systems were hydrated with 150 mM NaCl electrolyte. The all-atom CHARMM36 force field was used for protein, lipids, and ions, with TIP3P water (65⇓⇓–68). The MD trajectories were analyzed with Visual Molecular Dynamics (VMD) software (69).

All simulations were performed using GROMACS 5.0.6 (70). The starting systems were energy-minimized for 5,000 steepest descent steps and equilibrated initially for 500 ps of MD in a canonical (NVT) ensemble and later for 8 ns in an isothermal-isobaric (NPT) ensemble under periodic boundary conditions. During equilibration, the restraints on the positions of nonhydrogen protein atoms of initially 4,000 kJ⋅mol−1⋅nm2 were gradually released. Particle-mesh Ewald summation (71) with cubic interpolation and a 0.12-nm grid spacing was used to treat long-range electrostatic interactions. The time step was initially 1 fs, and was then increased to 2 fs during the NPT equilibration. The LINCS algorithm (72) was used to fix all bond lengths. Constant temperature was set with a Berendsen thermostat (73), with a coupling constant of 1.0 ps. A semiisotropic Berendsen barostat was used to maintain a pressure of 1 bar. During the production run, the Berendsen thermostat and barostat were replaced by a Nosé–Hoover thermostat (74) and a Parrinello–Rahman barostat (75). Analysis was carried out on unconstrained simulations.

Outward-facing state.

Initially, the outward-facing structure was embedded in a bilayer of 68 palmitoyl oleoyl phosphatidyl-glycerol (POPG), 102 palmitoyl oleoyl phosphatidic acid (POPA), and 168 palmitoyl oleoyl phosphatidyl-inositol (POPI) lipids. A 1,200-ns unconstrained production simulation was carried out for the system at 358 K. To construct an archaeal lipid bilayer, a snapshot from the simulation after 500 ns was used and the lipids were replaced by their archaeal counterparts (i.e., POPA by DPA, POPG by DPG, and POPI by DPI). After the equilibration phase, the archaeal lipid-embedded PfMATE was simulated for 1.5 μs at 373 K.

Inward-facing state.

The inward-facing structure was embedded in a bilayer of 68 POPG, 102 POPA, and 170 POPI lipids. A 450-ns unconstrained production simulation was carried out for the system at 358 K. To assess the putative sodium-binding site, a sodium ion was placed near Asp41/Asp148. After equilibration, a 250-ns unconstrained production simulation was carried out at 358 K.

Targeted MD simulation.

To explore the conformational dynamics during the alternation of the access state, we performed a targeted MD simulation starting from the inward-facing structure and targeting the outward-facing structure. For the targeted MD, we used the PLUMED v2.1 (76) patch in GROMACS 5.0.6. A bias potential acting on the Cα RMSD pushed the initial structure, the membrane embedded inward-facing structure of PfMATE, toward the target structure, the outward-facing structure. The targeted MD run was 50 ns.

Drug Susceptibility Test.

MIC tests were performed in E. coli KAM32 (ΔacrB, ΔydhE, and hsdΔ5) cells, which lack the genes encoding the major multidrug efflux pumps AcrB and YdhE, and the restriction system hsd (77). To determine the MIC value, a series of agar plates or culture tubes was prepared with a twofold increase of the concentrations of the toxic compound. E. coli KAM32/pBAD-A2-PfMATE and E. coli KAM32/pBAD-A2 (negative control) were exposed to different antibiotics and toxic compounds in LB medium as well as Müller Hinton (MH) broth. Briefly, 1 mL of LB or MH medium containing 0.02% (wt/vol) arabinose as an inducer and screening xenobiotics was inoculated with 105–106 cells, which were already induced with arabinose for 3 h to produce the target protein. Inoculated media were incubated at 37 °C overnight. In the case of the MIC tests on the agar plates, 10-fold–diluted solutions were spotted on the LB agar plates supplemented with 50 μg/mL carbenicillin, tested compound, and an expression inducer (0.02% arabinose). After 16 h of incubation, bacterial growth was analyzed visually. The MIC value was defined as the lowest concentration of each compound, which prevented visible growth. E. coli strains BW25113A (ΔacrAB, ΔtolC, ΔydhE, ΔmdfA, and ΔemrE), BW25113B (ΔacrAB, ΔydhE, and ΔemrE), and BW25113C (ΔacrB), as well as E. coli C43(DE3) ΔacrAB and E. coli C41(DE3) ΔacrAB, were also used for the MIC tests. Control cells containing an empty plasmid (pBAD) were also screened along with the test cells containing expression plasmid (pBAD-PfMATE). All experiments were repeated at least three times independently.

Data Availability.

The structure factors and coordinates for the inward- and outward-facing states obtained by the LCP method were deposited under PDB ID codes 6FHZ and 6GWH, respectively. The crystallographic data corresponding to the outward-facing structures obtained by the vapor diffusion method were deposited under PDB ID codes 6HFB and 4MLB.

Acknowledgments

We thank Barbara Rathmann and Yvonne Thielmann for help with the crystallization trials; the staff at the X10SA beamline (SLS, Villigen, Switzerland) for assistance at the synchrotron; Glycon Biochemicals for synthesizing octyl-β-d-selenoglucoside upon request; Ben Luisi for providing the C43(DE3) ∆acrAB strain and Klaas Martinus Pos for providing the C41(DE3) ∆acrAB strain; Renate Richau, Thomas Hader, and Konrad Eichinger for assistance with P. furiosus cultivation; Harald C. Köfeler, Martin Trötzmüller, and Christine Pein for lipidomics experiments; and Ulrich Ermler and Hao Xie for discussions. This work was supported by the Max Planck Society, the Deutsche Forschungsgemeinschaft (Cluster of Excellence Macromolecular Complexes Frankfurt), and CRC807 (Transport and Communication across Biological Membranes). Lipidomics experiments were supported by the Austrian Ministry for Education, Science and Research (JPI-HDHL Project BMWFW-10.420/0005-W/V/3c/2017 and HSRSM Grant Omics Center Graz, BioTechMed-Graz).

Footnotes

  • ↵1To whom correspondence may be addressed. Email: schara.safarian{at}biophys.mpg.de or hartmut.michel{at}biophys.mpg.de.
  • 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 the original 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 crystallization leading to the 6HFB and 4MLB structure determination; V.N.M. performed the functional characterization 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 manuscript with contributions from G.H. and H.M.; H.M. suggested experiments; G.H. and H.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.

  • 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 Protein Data Bank (PDB), www.pdb.org (PDB ID codes 6FHZ and 6GWH, respectively). The crystallographic data corresponding to the outward-facing structures obtained by the vapor diffusion method were deposited in the PDB (PDB ID codes 6HFB and 4MLB).

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

Published under the PNAS license.

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Inward-facing conformation of a multidrug resistance MATE family transporter
Sandra Zakrzewska, Ahmad Reza Mehdipour, Viveka Nand Malviya, Tsuyoshi Nonaka, Juergen Koepke, Cornelia Muenke, Winfried Hausner, Gerhard Hummer, Schara Safarian, Hartmut Michel
Proceedings of the National Academy of Sciences Jun 2019, 116 (25) 12275-12284; DOI: 10.1073/pnas.1904210116

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Inward-facing conformation of a multidrug resistance MATE family transporter
Sandra Zakrzewska, Ahmad Reza Mehdipour, Viveka Nand Malviya, Tsuyoshi Nonaka, Juergen Koepke, Cornelia Muenke, Winfried Hausner, Gerhard Hummer, Schara Safarian, Hartmut Michel
Proceedings of the National Academy of Sciences Jun 2019, 116 (25) 12275-12284; DOI: 10.1073/pnas.1904210116
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