Gram-negative bacteria, such as Escherichia coli, expel toxic chemicals through tripartite efflux pumps that span both the inner and outer membrane. The three parts are an inner membrane, substrate-binding transporter; a membrane fusion protein; and an outer-membrane-anchored channel. The fusion protein connects the transporter to the channel within the periplasmic space. A crystallographic model of this tripartite efflux complex has been unavailable because co-crystallization of the various components of the system has proven to be extremely difficult. We previously described the crystal structures of both the inner membrane transporter CusA1 and the membrane fusion protein CusB2 of the CusCBA efflux system3,4 of E. coli. Here we report the co-crystal structure of the CusBA efflux complex, showing that the transporter (or pump) CusA, which is present as a trimer, interacts with six CusB protomers and that the periplasmic domain of CusA is involved in these interactions. The six CusB molecules seem to form a continuous channel. The affinity of the CusA and CusB interaction was found to be in the micromolar range. Finally, we have predicted a three-dimensional structure for the trimeric CusC outer membrane channel and developed a model of the tripartite efflux assemblage. This CusC3–CusB6–CusA3 model shows a 750-kilodalton efflux complex that spans the entire bacterial cell envelope and exports Cu i and Ag i ions.
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This work is supported by National Institutes of Health grants R01GM074027 (E.W.Y.), R01GM086431 (E.W.Y.), R01GM081680 (R.L.J.) and R01GM072014 (R.L.J.). This work is based on research conducted at the Northeastern Collaborative Access Team beamlines of the Advanced Photon Source, supported by award RR-15301 from the National Center for Research Resources at the National Institutes of Health. Use of the Advanced Photon Source is supported by the US Department of Energy, Office of Basic Energy Sciences, under contract DE-AC02-06CH11357.
The authors declare no competing financial interests.
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Su, C., Long, F., Zimmermann, M. et al. Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli. Nature 470, 558–562 (2011) doi:10.1038/nature09743
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