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 (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs 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 MFPs, 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 an MFP in the resistance–nodulation–division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.

Introduction

Silver is a heavy metal with relatively high toxicity to prokaryotes. Ionic silver exhibits antimicrobial activity against a broad range of microorganisms and is used widely as an effective antimicrobial agent to combat pathogens.1, 2 Copper, although required in trace amounts for bacterial growth, is highly toxic even at low concentrations.³ Thus, both silver and copper are well-known bactericides, and their biocidal effects have been used for centuries. It has been shown that silver and copper ions effectively eliminate Legionella in drinking water pipelines.⁴ Silver and copper ions are capable of penetrating biofilms that build up in hospital plumbing, destroying entrenched Legionella and other pathogenic organisms.4, 5 In addition, silver cations are commonly employed in treating patients with burns, wounds, eye infections, and ulcers.¹ To date, the antimicrobial uses of ionic copper have been expanded to include fungicides, antifouling paints, antimicrobial medicines, and antiseptics‡. Because of the widespread use of silver and copper as antimicrobial agents, the presence of silver- and copper-resistant bacterial strains appears to be on the rise.1, 2, 6, 7, 8

Bacteria, such as Escherichia coli, have developed various mechanisms to overcome toxic environments that are unfavorable to their survival. One important strategy that bacteria use to subvert toxic compounds, including toxic metal ions such as Ag⁺ and Cu⁺, is the expression of membrane efflux transporters that recognize and actively export these compounds out of bacterial cells, thereby allowing them to survive in extremely toxic conditions. In Gram-negative bacteria, efflux systems of the resistance–nodulation–division (RND) family play major roles in the intrinsic and acquired tolerance of antibiotics and toxic compounds.9, 10 As a Gram-negative bacterium, E. coli contains seven different RND efflux transporters. Six of these transporters, including AcrB, AcrD, AcrF, MdtB, MdtC, and YhiV, are multidrug efflux pumps. They belong to the hydrophobic and amphiphilic efflux RND (HAE-RND) protein family.⁹ E. coli consists of only one heavy-metal efflux RND (HME-RND) transporter, CusA, which specifically recognizes and confers resistance to Ag(I) and Cu(I) ions.11, 12

Typically, an RND transporter works in conjunction with a periplasmic component, belonging to the membrane fusion protein (MFP) family,13, 14 and an outer-membrane channel to form a functional protein complex.¹⁵ The resulting tripartite efflux system spans the inner and outer membranes of Gram-negative bacterium to export substrates directly out of the cell.¹⁵ For the CusA inner-membrane transporter, it interacts with the periplasmic MFP CusB and the outer-membrane channel CusC to form the CusABC tripartite efflux complex.11, 12 Heavy-metal efflux by CusABC is driven by proton import. This process is catalyzed through the inner-membrane transporter CusA.

Among all known RND family of transporters, the E. coli AcrB16, 17, 18, 19 and Pseudomonas aeruginosa MexB²⁰ HAE-RND pumps are the only two membrane proteins that have been crystallized. These proteins span the entire width of the inner membrane and protrude approximately 70 Å into the periplasm. The crystal structures of the outer-membrane channels E. coli TolC and P. aeruginosa OprM have also been determined.21, 22 TolC is anchored in the outer membrane and forms a 100-Å-long periplasmic α-helical tunnel.²¹ The P. aeruginosa OprM channel possesses a similar elongated α-helical tunnel that projects into the periplasmic space.²² Recently, two structures of the periplasmic MFPs, E. coli AcrA²³ and P. aeruginosa MexA,^24–26 associated with the HAE-RND transporters have been solved. The structures suggest that these two periplasmic proteins are folded into elongated secondary structures that consist of ∼ 47-Å-long α-hairpin domain, presumably interacting with the α-helical tunnels of their corresponding outer-membrane channels. Further, the N- and C-terminal ends of these MFPs are thought to contact their respective inner-membrane transporters, creating a functional complex that spans both membranes.

Currently, no structural information is available for any components of the HME-RND tripartite efflux complex. Presumably, the three components of the HME-RND system form a tripartite complex that resembles the AcrAB–TolC complex. Different from the HAE-RND family, members of the HME-RND family are highly substrate specific, with the ability to differentiate between monovalent and divalent ions. As an initial step to examine the mechanisms used by the CusABC efflux system to facilitate Ag(I) and Cu(I) ion recognition and extrusion, we here describe the crystal structures of the periplasmic MFP CusB in the absence and presence of these metal ions.