Crystal Structure of the Membrane Fusion Protein CusB from Escherichia coli

Abstract

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein that is critical for substrate transport. We here present the x-ray structures of the CusB membrane fusion protein from the copper/silver efflux system of E. coli. This is the first structure of any membrane fusion proteins associated with heavy-metal efflux transporters. CusB bridges the inner membrane efflux pump CusA and outer membrane channel CusC to mediate resistance to Cu+ and Ag+ ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly β-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of membrane fusion proteins, the α-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N and C-termini of CusB form the first β-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu+ and Ag+ were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of a membrane fusion protein in the resistance-nodulation-division efflux system, and provide evidence that this protein specifically interacts with transported substrates.

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.…”

“…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.…”

“…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.…”

Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex

“…The crystal structure of CusB (pdb code: 3H9I), comprising approximately 80 per cent of the protein (residues 89-385), was originally determined to a resolution of 3.40 Å [36]. The structure revealed that the asymmetric unit of the crystal consists of two protomers.…”

“…This domain is located directly above the outer-leaflet of the inner membrane. Using lysine-lysine cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/ MS), it has been found that this domain specifically interacts with the periplasmic domain of the CusA efflux pump [36]. Overall, domain 1 is a b-barrel domain, consisting of six b-strands, with the N-terminal end forming one of the b-strands while the C-terminus of the protein constitutes the remaining five strands (figure 1a).…”