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

Abstract

Gram-negative bacteria, such as Escherichia coli, expel toxic chemicals via tripartite efflux pumps spanning both the inner and outer membranes. The three parts are: 1) a membrane fusion protein connecting 2) a substrate-binding inner membrane transporter to 3) an outer membrane-anchored channel in the periplasmic space. A crystallographic model of this tripartite efflux complex has been unavailable simply because co-crystallization of different components of the system has proven to be extremely difficult. We previously described the crystal structures of both the inner membrane transporter CusA1 and membrane fusion protein CusB2 of the CusCBA efflux system3,4 from E. coli. We here report the co-crystal structure of the CusBA efflux complex, revealing the trimeric CusA efflux pump interacts with six CusB protomers at the upper half of the periplasmic domain. These six CusB molecules form a channel extending contiguously from the top of the pump. The affinity of the CusA and CusB interaction was found to be in the micromolar range. Finally, we predicted a three-dimensional structure of the trimeric CusC outer membrane channel, and develop a model of the tripartite efflux assemblage. This CusC3-CusB6-CusA3 model presents a 750 kDa efflux complex spanning the entire bacterial cell envelope to export Cu(I)/Ag(I) ions.

Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

“…In the "switch" model, copper loading of CusB induces a conformational change (22), which allows CusB to bind to CusA and open the entry site on the CusA pump, which is known to be located in its periplasmic cleft (26,34). Although both models predict that either CusB deletion, or metal-site mutagenesis should lead to copper sensitive strains-an expectation that has been experimentally verified (22,23)-definitive evidence for or against either model has been lacking and is compounded by the fact that the copper-binding N-terminal domain of CusB is disordered in crystal structures of the isolated protein (27), or its complex with CusA (29). Existing evidence, although weak, has leaned more toward the switch model, based mainly on projections of the distance of the CusB metal-binding site from the CusA periplasmic cleft (30), and analogy to structural data on other metal resistance RND adaptor proteins such as ZneB of Cupriavidus metallodurans CH34.…”

“…Although these structures have provided enormous insight into possible modes of metal efflux (30,31), the dynamic mechanism of periplasmic metal detoxification by Cus is still undetermined. This is in part due to disorder in the crystal structure of the metalbinding site of CusB (27,29) and the difficulty of creating an in vitro scenario that includes CusA, CusB, and CusF in biologically relevant conditions. The purpose of our study was to create such a scenario and to determine the method of activation of the CusA pump, thereby defining the exact roles of CusF and CusB in periplasmic Ag and Cu efflux.…”

“…Crystal structures of CusA (26), CusB (27), and the outer membrane protein CusC (28) have been reported in recent years together with a structure of the CusBA complex (29), revealing a stoichiometry of 2:1:1 for the B:A:C components. Although these structures have provided enormous insight into possible modes of metal efflux (30,31), the dynamic mechanism of periplasmic metal detoxification by Cus is still undetermined.…”

Crystal structure of the transcriptional regulator Rv1219c of Mycobacterium tuberculosis

“…These three methionines form a triad, similar to the periplasmic heavy metal binding site of the CusA efflux pump. [29][30][31] It is possible that these three methionines may cooperate to create a metal binding site within the multidrug binding cavity. The crystal structures of Rv1219c bound with a variety of ligands will be crucial for further understanding of how this regulator recognizes multiple antimicrobials.…”

“…Each subunit of Rv1219c is composed of 10 helices (a1-a10 and a1 0 -a10 0 , respectively). The helices of Rv1219c are designated numerically from the N-terminus as a1 (7-23), a2 (29)(30)(31)(32)(33)(34)(35)(36), a3 (40-47), a4 (50-74), a5 (78-86), a6 (92-104), a7 (108-130), a8 (139-160), a9 (166-188), and a10 (194)(195)(196)(197)(198)(199)(200)(201)(202)(203)(204)(205)(206). In this arrangement, the smaller N-terminal domain includes helices a1 through a3 and the N-terminal end of a4 (residues 50-60), with a2 and a3 forming a typical helix-turn-helix motif.…”