Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli

Abstract

Gram-negative bacteria expel toxic chemicals through tripartite efflux pumps spanning both the inner and outer membranes. A crystallographic model of this tripartite efflux complex has been unavailable because co-crystallization of different components of the system has proved extremely difficult. The X-ray crystal structure of the CusA/CusB co-complex from Escherichia coli has now been determined. The structure reveals that the trimeric CusA efflux pump interacts with six CusB protein molecules at the upper half of the periplasmic domain, and the predicted structure of the trimeric CusC channel was used to develop a model of the tripartite efflux complex. Gram-negative bacteria expel toxic chemicals via tripartite efflux pumps spanning both the inner and outer membranes. A crystallographic model of this tripartite efflux complex has been unavailable because co-crystallization of different components of the system has proven to be extremely difficult. The X-ray crystal structure of CusA of the CusCBA tripartite efflux system from Escherichia coli has been reported previously, and here the X-ray crystal structure of the CusBA co-complex is reported. The structure reveals that the trimeric CusA efflux pump interacts with six CusB protein molecules at the upper half of the periplasmic domain, and the predicted structure of the trimeric CusC channel was used to develop a model of for the tripartite efflux complex. 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.