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  • br Results br Discussion We have reported the crystal struct

    2021-10-12


    Results
    Discussion We have reported the crystal structures of VcmN, the high-resolution structures of this MATE transporter from a pathogenic bacterium. The structures display distinct conformations of TM1, as a consequence of their unique hydrogen-bonding networks. We interpret this result to suggest that the transport Cy3 maleimide (non-sulfonated) sale of VcmN involves the rearrangement of the hydrogen-bonding network in the N-lobe and the bending of TM1, similar to other members of the DinF subfamily. Based on these findings, we propose a common working model for the transport mechanism of the bacterial and archaeal H+-coupled DinF transporters (Figure 4). In VcmN, the TM1 helix, which is deprotonated at Asp35, adopts the straight conformation favoring a substrate-binding-competent state (Figure 4A). Subsequent H+ binding to the conserved Asp35 in TM1 causes the rearrangement of the hydrogen-bonding network around Asp35, which in turn induces the bending of TM1. As a result, the substrate-binding pocket in the N-lobe shrinks, thereby preventing the re-binding of the extruded substrate (Figure 4B). In this mechanism, the binding of the substrate and the H+ to the transporter is mutually exclusive, which is consistent with an antiport mechanism for MATE transporters.
    STAR★Methods
    Acknowledgments We thank T. Nishizawa, H. Nishimasu, T. Nakane, and M. Fukuda for useful discussions, and K. Hirata and K. Yamashita, and the beamline staff members at BL32XU Cy3 maleimide (non-sulfonated) sale of SPring-8, for technical support with data collection. We also thank K. Ito for providing the acrB gene knockout E. coli strains and R. Taniguchi, Y. Lee and H.E. Kato for technical assistance. The X-ray diffraction experiments were performed at SPring-8 BL32XU (proposal nos. 2012B1146, 2013A1128, 2013A1168, 2014A1061, and 2014A1116), with the approval of RIKEN, and at Diamond Light Source I24. We thank S. Newstead for assistance in accessing Diamond Light Source I24. This work was supported by the Platform for Drug Discovery, Informatics, and Structural Life Science, funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), by a maximum sustainable yield (MSY) Grant-in-Aid for Scientific Research (S) (24227004) and a Grant-in-Aid for Scientific Research (B) (25291011) from the Japan Society for the Promotion of Science (JSPS) to O.N. and R.I., respectively, and by a Grant-in-Aid for JSPS Fellows to T. Kusakizako (26a9020). H.S.M. and D.P.C. were supported by a grant from the National Institute of General Medical Sciences (R01GM077659).