Visualizing the nonlinear changes of a drug-proton antiporter from inward-open to occluded state

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Highlights

  • Visualizing dynamic alternating access cycle is important for understanding the transport mechanism of MFS transporters.

  • Crystal structure of SotB in complex with IPTG presents a substrate-bound occlude conformation.

  • Crystal structures of SotB in different conformations.

  • Four structures comparison reveals the nonlinear conformational changes from an inward-open to an occluded state.

Abstract

Drug-proton antiporters (DHA) play an important role in multi-drug resistance, utilizing the proton-motive force to drive the expulsion of toxic molecules, including antibiotics and drugs. DHA transporters belong to the major facilitator superfamily (MFS), members of which deliver substrates by utilizing the alternating access model of transport. However, the transport process is still elusive. Here, we report the structures of SotB, a member of DHA1 family (TCDB: 2.A.1.2) from Escherichia coli. Four crystal structures of SotB were captured in different conformations, including substrate-bound occluded, inward-facing, and inward-open states. Comparisons between the four structures reveal nonlinear rigid-body movements of alternating access during the state transition from inward-open to occluded conformation. This work not only reveals the conformational dynamics of SotB but also deepens our understanding of the alternating access mechanism of MFS transporters.

Introduction

The major facilitator superfamily (MFS) is the largest family of secondary transporters, which includes thousands of integral membrane transporters in extremely diverse organisms [[1], [2], [3]]. The MFS transporters share similar structural folds and deliver a wide variety of substrates by three different transport patterns: uniport, symport, and antiport [[4], [5], [6]]. The MFS-fold transporters share a common 12 transmembrane (TM) helixes, which is divided into two six-helix bundles (N and C domains) connected by a cytosolic loop region [6,7]. The distinct structural conformations of several MFS transporters have been determined by X-ray crystallography [5,[8], [9], [10], [11]]. Based on the structures of three major conformations (inward-facing, occluded, and outward-facing), the global rocker-switch rearrangement model was proposed to explain the alternating access mechanism of MFS transporters [5,6]. In addition, GLUTs, which function as uniporters or proton symporters with MFS folds, undergo local conformational changes of the gating helix upon glucose binding [[12], [13], [14]] and this local conformational movement was perceived as a structural prerequisite [7]. It is not known whether MFS antiporters require similar local movements for transport.

Drug-proton antiporters (DHA) from the MFS family constitute a major portion of multidrug-resistance transporters and utilize the proton-motive force to drive the expulsion of toxic compounds [[15], [16], [17], [18]]. To understand the molecular mechanism of proton coupling and antiport, several biochemical and structural investigations have been conducted [[19], [20], [21], [22]]. In antiporters, substrate binding and protonation are often found to compete with each other [23]. Substrate binding-induced deprotonation is thought to trigger the conformational change from inward-facing to outward-facing. As with symporters, there are three major states (inward facing/open, occluded, and outward facing/open) in the alternating access cycle of antiporters [24]. Determining the structures of antiporters in different conformations is one of the primary methods being used to understand the mechanism of alternating access. However, although progress on the structural elucidation of drug-proton antiporters has been made, available structures are not sufficient to explain the process in detail. There are currently only a few structures available from antiporters GlpT [8], YajR [11], EmrD [21] and MdfA [20,25]. Among them, the structure of MdfA, a well-studied prototype multidrug-resistance transporter [26], has been solved in two different conformations, including substrate-bound inward-facing and outward-open [20,25]. Biochemical studies of MdfA confirmed competition between substrate and protons in secondary multidrug transport [23]. However, the structural basis of the alternating access mechanism of DHA transport remains to be elucidated.

In Escherichia coli (E. coli), SotB is classified as a drug efflux transporter and into the 12 TM domain drug-resistance proton driven antiporter (DHA1) subfamily [27]. Previously, biochemical studies showed that SotB prevents accumulation of arabinose and IPTG in cytoplasm [28,29]. In addition, SotB has an important conserved motif (Motif C), also called the antiporter motif, which is located within the fifth transmembrane helix [30,31].

To better understand the alternating access of DHA antiporter, we conducted a comprehensive structural investigation on SotB. We reported four crystal structures of SotB: SotB-IPTG at 3.05 Å, substrate-free SotB at 3.4 Å, SotBH115A at 3.5 Å, and SotBH115N at 2.65 Å resolutions, respectively. Structural comparisons between the four structures reveal the complete mechanism of alternating access from inward-open to occluded states. The observed intermediate states of SotB show the rigid-body movement of the N and C domains and the nonlinear motion path, providing insights into the rigid conformational changes induced by substrate binding to DHA transporters.

Section snippets

Expression and purification

The cDNA of full-length SotB from E. coli strain BL21 (DE3) was subcloned into modified pET15b with additional drICE cutting site (DEVDA). The mutants of SotB were generated by standard two-step PCR. Overexpression of SotB was induced in E. coli C43 (DE3) by 0.3 mM isopropyl β-D-thiogalactoside (IPTG) at 22 °C overnight when the cell density reached to an OD600 of 1.0. After induction, cells were harvested and homogenized in lysis buffer (25 mM Tris-HCl pH 8.0, 20 mM IPTG, 150 mM NaCl, and 1 mM

Structure of wild-type SotB in complex with IPTG

The wild-type full-length protein of SotB in complex with IPTG was crystallized in the space group P212121 using the hanging-drop vapor diffusion method. The structure was determined by molecular replacement using the predicted model of SotB generated by the Robetta server [35] and refined to 3.05 Å (Table S1). Since previous studies have suggested that SotB is able to export l-arabinose and IPTG [28,29], SotB was crystallized in a buffer containing 20 mM IPTG. After assignment of the SotB

Discussion

Currently, precise structure-by-structure prediction is challenging. However, the predicted structures of several proteins with specific folds, such as MFS fold and LeuT fold, are able to provide useful information for further investigation. In this study, we solved the structures of SotB by molecular replacement using the predicted structure as the search model, although SotB exist the multiple conformations and has less than 20% sequence identity with the solved structure.

We presented four

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgments

We thank staff at Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U1, BL18U, and BL19U1 for on-site assistance. This work was supported by funds from National Key R&D Program of China (grant number 2016YFA0502700), National Natural Science Foundation of China (grant number 31971132).

References (44)

  • V.S. Reddy et al.

    The major facilitator superfamily (MFS) revisited

    FEBS J.

    (2012)
  • N. Yan

    Structural biology of the major facilitator superfamily transporters

    Annu. Rev. Biophys.

    (2015)
  • E.M. Quistgaard et al.

    Understanding transport by the major facilitator superfamily (MFS): structures pave the way

    Nat. Rev. Mol. Cell Biol.

    (2016)
  • D. Drew et al.

    Shared molecular mechanisms of membrane transporters

    Annu. Rev. Biochem.

    (2016)
  • Y. Huang et al.

    Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli

    Science

    (2003)
  • S. Dang et al.

    Structure of a fucose transporter in an outward-open conformation

    Nature

    (2010)
  • D. Jiang et al.

    Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A

    Proc. Natl. Acad. Sci. U. S. A.

    (2013)
  • D. Deng et al.

    Crystal structure of the human glucose transporter GLUT1

    Nature

    (2014)
  • D. Deng et al.

    Molecular basis of ligand recognition and transport by glucose transporters

    Nature

    (2015)
  • N. Nomura et al.

    Structure and mechanism of the mammalian fructose transporter GLUT5

    Nature

    (2015)
  • I.T. Paulsen et al.

    Proton-dependent multidrug efflux systems

    Microbiol. Rev.

    (1996)
  • C.F. Higgins

    Multiple molecular mechanisms for multidrug resistance transporters

    Nature

    (2007)
  • Cited by (0)

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    These authors contributed equally to this work.

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