Visualizing the nonlinear changes of a drug-proton antiporter from inward-open to occluded state Qingjie Xiao a, 1 , Bo Sun b, 1 , Yanxia Zhou a , Chen Wang a , Li Guo a , Jianhua He b, * , Dong Deng a, ** a From the Division of Obstetrics, Key Laboratory of Birth Defects and Related Disease of Women and Children of MOE, State Key Laboratory of Biotherapy, West China Second Hospital, Sichuan University, Chengdu, 610041, China b Shanghai Synchrotron Radiation Facility Science Center, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, 201204, China article info Article history: Received 19 November 2020 Accepted 24 November 2020 Available online xxx Keywords: Drug-proton antiporter SotB Crystal structure Multiple conformations Nonlinear change 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 trans- porters belong to the major facilitator superfamily (MFS), members of which deliver substrates by uti- lizing 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. © 2020 Elsevier Inc. All rights reserved. 1. Introduction The major facilitator superfamily (MFS) is the largest family of secondary transporters, which includes thousands of integral membrane transporters in extremely diverse organisms [1e3]. The MFS transporters share similar structural folds and deliver a wide variety of substrates by three different transport patterns: uniport, symport, and antiport [4e6]. 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 [5,8e11]. 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 alter- nating 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 [12e14] 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 [15e18]. To understand the molecular mechanism of proton coupling and antiport, several biochemical and structural in- vestigations have been conducted [19e22]. 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 * Corresponding author. ** Corresponding author. E-mail addresses: hejianhua@sinap.ac.cn (J. He), dengd@scu.edu.cn (D. Deng). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2020.11.096 0006-291X/© 2020 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (xxxx) xxx Please cite this article as: Q. Xiao, B. Sun, Y. Zhou et al., Visualizing the nonlinear changes of a drug-proton antiporter from inward-open to occluded state, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.11.096