Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell.1,2 The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions.3,4 No prior structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide important new structural information about the HME sub-family of RND efflux pumps. The structures suggest that the metal binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. Intriguingly, this cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal binding site, four methionine pairs - three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, utilizing these methionine pairs/clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.

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.

Neisseria gonorrhoeae is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in N. gonorrhoeae is the multiple transferrable resistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the mtrD gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation. IMPORTANCE Neisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD, encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.

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.