br Conflict of interest statement br Acknowledgments This wo

Conflict of interest statement
Acknowledgments This work was funded in part by a grant from the National Institutes of Health, NIDDK award #DK61425 (to PWS).
Introduction The efflux of toxic compounds from the cell by multidrug exporters is an important mechanism for cellular homeostasis and survival (Blair et al., 2014, Du et al., 2015). One family of multidrug exporters, the multidrug and toxic Hydroxysafflor yellow A synthesis extrusion (MATE) family, actively transports xenobiotics and metabolic organic cations, using an electrochemical Na+ or H+ gradient across the membrane (Brown et al., 1999, He et al., 2004, Jin et al., 2014). MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, highlighting their clinical importance (Kaatz et al., 2005, McAleese et al., 2005). MATE transporters are highly conserved among bacteria, archaea, and eukarya, and are classified into three branches: the NorM, DinF, and eukaryotic subfamilies (Omote et al., 2006). Over the last decade, structural and biochemical analyses of MATE transporters have been intensively pursued (Miyauchi et al., 2017, Tanaka et al., 2013, Tanaka et al., 2017, He et al., 2010, Lu et al., 2013a, Lu et al., 2013b, Mousa et al., 2016, Radchenko et al., 2015). To date, the crystal structures of five prokaryotic MATE transporters have been reported: Vibrio cholerae NorM subfamily (NorM-VC) (He et al., 2010) simultaneously coupled to the Na+ and H+ gradients (Jin et al., 2014), two Na+-driven MATE transporters (Neisseria gonorrhoeae NorM [NorM-NG] [Lu et al., 2013a] and Escherichia coli DinF subfamilies [ClbM] [Mousa et al., 2016]), and two H+-driven MATE transporters (Pyrococcus furiosus DinF [PfMATE] [Tanaka et al., 2013] and Bacillus halodurans DinF subfamilies [DinF-BH] [Lu et al., 2013b, Radchenko et al., 2015]). These structures revealed that the basic architecture of the prokaryotic MATE transporters consists of a unique topological arrangement of 12 transmembrane (TM) helices, divided between an N-lobe (TM1–TM6) and a C-lobe (TM7–TM12). The arrangement of the helices within each lobe leads to the formation of cavities, which have been proposed to bind substrates. The substrate-bound and cation-bound structures have provided insights into the details of substrate recognition and implications for the transport mechanism by prokaryotic MATE transporters. Despite this remarkable progress on the structural front, the substrate transport mechanism by H+-driven DinF subfamily transporters has remained controversial. Our previous structural and mutational analyses of H+-driven PfMATE suggested that H+ binding to the conserved Asp41 in TM1 allosterically induces the bending of TM1, which in turn reduces the volume of the N-lobe cavity where substrates bind, presumably releasing the bound substrate. On the basis of this work, we proposed that the proton-induced collapse of the substrate-binding pocket would be the last step of the substrate extrusion mechanism (Tanaka et al., 2013, Nishima et al., 2016). In contrast, structural and biochemical analyses of H+-driven DinF-BH suggested that the H+ and substrates share the conserved Asp40 in TM1 (corresponding to Asp41 of PfMATE) as the binding site, implying that H+ binding directly triggers the release of the bound substrate (Lu et al., 2013b). Furthermore, because the structure of the DinF-BH D40N mutant, which is presumed to mimic the protonation of Asp40, adopted the TM1-straight conformation similar to the wild-type, it was proposed that the conformational change of TM1 does not necessarily occur during the transport cycle of DinF-BH (Radchenko et al., 2015). To address the controversy surrounding the H+-coupled substrate transport mechanism, and investigate the extent to which the structural changes in TM1 are conserved, we determined the crystal structures of VcmN, an H+-driven MATE transporter from Vibrio cholerae, in distinct conformations. A structural comparison revealed a conformational change of TM1, which is putatively associated with protonation and involves the rearrangement of a hydrogen-bonding network in the N-lobe. This conformational change was impaired by the mutation of the critical Asp35 in TM1, consistent with the notion that the conserved Asp is critical for the bending of TM1. Based on these results, we propose common intermediates in the transport mechanism shared among the bacterial and archaeal H+-driven MATE transporters.