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 beta-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the alpha-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 beta-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.

Figure 1

Stereo view of the experimental electron density map at a resolution of 3.8 Å. (a) The electron density map contoured at 1.2σ is in gray. The C^α traces of molecules A and B of CusB are in orange and green, respectively. (b) Representative section of the electron density in the second domain of CusB. The electron density (colored blue) is contoured at the 1.2σ level and superimposed with the final refined model (orange, carbon; red, oxygen; blue, nitrogen).

Figure 1

Stereo view of the experimental electron density map at a resolution of 3.8 Å. (a) The electron density map contoured at 1.2σ is in gray. The C^α traces of molecules A and B of CusB are in orange and green, respectively. (b) Representative section of the electron density in the second domain of CusB. The electron density (colored blue) is contoured at the 1.2σ level and superimposed with the final refined model (orange, carbon; red, oxygen; blue, nitrogen).

Figure 2

Crystal structure of the CusB membrane fusion protein. The structure can be divided into four distinct domains. Domain 1 is formed by the N and C-termini and is located above the inner membrane. The loops between Domains 2 and 3 appear to form an effective hinge to allow the molecule to shift from an open conformation to a more compact structure. Domain 4 is folded into an anti-parallel, three-helix bundle, which is thought to be located near the outer membrane.

Figure 3

Structural comparison of the membrane fusion proteins. (a) Superimposition of the crystal structures of CusB (orange) and MexA (purple). (b) Superimposition of Domain 1 of CusB (orange) with the membrane proximal domain of MexA (purple). (c) Superimposition of Domain 2 of CusB (orange) with the β-barrel domain of MexA (purple). (d) Superimposition of Domain 3 of CusB (orange) with the lipoyl domain of MexA (purple).

Figure 3

Structural comparison of the membrane fusion proteins. (a) Superimposition of the crystal structures of CusB (orange) and MexA (purple). (b) Superimposition of Domain 1 of CusB (orange) with the membrane proximal domain of MexA (purple). (c) Superimposition of Domain 2 of CusB (orange) with the β-barrel domain of MexA (purple). (d) Superimposition of Domain 3 of CusB (orange) with the lipoyl domain of MexA (purple).

Figure 3

Structural comparison of the membrane fusion proteins. (a) Superimposition of the crystal structures of CusB (orange) and MexA (purple). (b) Superimposition of Domain 1 of CusB (orange) with the membrane proximal domain of MexA (purple). (c) Superimposition of Domain 2 of CusB (orange) with the β-barrel domain of MexA (purple). (d) Superimposition of Domain 3 of CusB (orange) with the lipoyl domain of MexA (purple).

Figure 3

Structural comparison of the membrane fusion proteins. (a) Superimposition of the crystal structures of CusB (orange) and MexA (purple). (b) Superimposition of Domain 1 of CusB (orange) with the membrane proximal domain of MexA (purple). (c) Superimposition of Domain 2 of CusB (orange) with the β-barrel domain of MexA (purple). (d) Superimposition of Domain 3 of CusB (orange) with the lipoyl domain of MexA (purple).

Figure 4

Comparison of the two conformations of CusB observed in the crystal. (a) Superposition of Domains 1 + 2 of molecule A onto Domains 1 + 2 of molecule B, displaying an ~21° overall shift of the three-helix bundle of Domain 4. (b) Superposition of Domains 3 + 4 of molecule A onto Domains 3 + 4 of molecule B, displaying an overall shift of the β-strands of Domain 1 by ~23°.

Figure 4

Comparison of the two conformations of CusB observed in the crystal. (a) Superposition of Domains 1 + 2 of molecule A onto Domains 1 + 2 of molecule B, displaying an ~21° overall shift of the three-helix bundle of Domain 4. (b) Superposition of Domains 3 + 4 of molecule A onto Domains 3 + 4 of molecule B, displaying an overall shift of the β-strands of Domain 1 by ~23°.

Figure 5

Cu⁺ and Ag⁺ binding sites of molecule A of CusB. Cu⁺ and Ag⁺ ions are represented by purple and green spheres, respectively. The overall locations of sites C1,C2 and A1 are circled. Anomalous difference Fourier maps are contoured at 4.6 σ, 4.0 σ and 4.2 σ for sites C1, C2 and A1, respectively. Anomalous peak heights for sites C1′, C2′ and A1′ in molecule B of CusB (not shown) were found to be 4.6 σ, 4.6 σ and 5.4 σ, respectively.

Figure 6

Specific interaction between CusA and CusB. The model of CusA (gray) was created based on protein sequence alignment and the crystal structure of AcrB. Mass spectral data suggest that the periplasmic domain of CusA specifically interacts with the N-terminus of CusB (light brown). Polypeptides α and β were in red and blue, respectively.