Cryo-EM Structures of CusA Reveal a Mechanism of Metal-Ion Export

Cryo-EM structure of the CusA trimer in the EBB state. (A) Ribbon diagram of the CusA trimer with one closed cleft “extrusion” state protomer and two open cleft “binding” state protomers viewed from the membrane plane with the “extrusion” and “binding” channels shown in yellow. (B) Ribbon diagram of the EBB CusA trimer viewed from the top of the periplasmic domain illustrating the channels (colored yellow) formed in the “binding” and “extrusion” states of the CusA protomers. (C) A cartoon displaying the conformation of the CusA trimer in the EBB form viewed from the top of the periplasmic domain. In panels A, B, and C, the one “extrusion” and two “binding” protomers of CusA are colored pink, green, and blue, respectively. The bound Cu(I) ions within the two “binding” protomers are represented as dark orange circles. (D) The binding site of the Cu(I) ion (dark orange sphere) within the open periplasmic cleft of the “binding” protomer (green). The bound Cu(I) ion is coordinated by residues M573, M623, E625, and M672 (gold sticks). The density of bound Cu(I) is shown in transparent light green.

Cryo-EM structure of the CusA trimer in the EEE state. (A) Ribbon diagram of the CusA trimer with three closed cleft “extrusion” state protomers viewed from the membrane plane with the “extrusion” channels shown in yellow. (B) Ribbon diagram of the EEE CusA trimer viewed from the top of the periplasmic domain illustrating the channels (colored yellow) formed in the “extrusion” states of the CusA protomers. (C) A cartoon displaying the conformation of the CusA trimer in the EEE form viewed from the top of the periplasmic domain. In panels A, B, and C, the three “extrusion” protomers of CusA are colored pink, green, and blue, respectively. (D) The Cu(I) binding site. No extra density representing bound Cu(I) is found within closed periplasmic cleft of this “extrusion” protomer (green). Residues M573, M623, E625, and M672 responsible for forming the Cu(I) binding site are in gold sticks.

Cryo-EM structure of the CusA trimer in the BBB state. (A) Ribbon diagram of the CusA trimer with three open cleft “binding” state protomers viewed from the membrane plane with the “binding” channels shown in yellow. (B) Ribbon diagram of the BBB CusA trimer viewed from the top of the periplasmic domain illustrating the channels (colored yellow) formed in the “binding” states of the CusA protomers. (C) A cartoon displaying the conformation of the CusA trimer in the BBB form viewed from the top of the periplasmic domain. In panels A, B, and C, the three “binding” protomers of CusA are colored pink, green, and blue, respectively. (D) The binding site of the Cu(I) ion (dark orange sphere) within the open periplasmic cleft of the “binding” protomer (pink). The bound Cu(I) ion is coordinated by residues M573, M623, E625, and M672 (gold sticks). The density of bound Cu(I) is shown in transparent light green.

The proton relay and methionine relay networks. (A) The proton relay network of the CusA heavy-metal efflux pump. (Left) The binding state of CusA (light blue). In this conformational state, the “proton sweeper” K984 was found to have two alternate rotamer conformations. One rotamer forms a hydrogen bond (yellow dashed line) with D405. D405 also forms hydrogen bonds with E939 and T987. The other rotamer of K984 is positioned away from D405 and forms a hydrogen bond with S1027. Black arrows represent the motion of the TM helices when they shift to the extrusion state. The densities of residue side chains (D405, K984, E939, and T987), which form the proton relay network are represented as a gray transparent surface. (Right) The extrusion state of CusA (light pink). In this conformation, K984 is swung away from D405 oriented to participate in a dipole-dipole interaction with M944. Superimposition of the CusA TM domain of the binding and extrusion states gives rise to an r.m.s.d. of 1.2 Å. (B) The methionine relay network of the CusA heavy-metal efflux pump. (Top left) Ribbon diagram of the binding state protomer of CusA with the channel for metal ion transport (yellow) and methionine residues (dark gold sticks) participating in metal transport viewed from the transmembrane region of the “binding” protomer of the EEB structure with the channel spanning from the cytosol into the periplasmic cleft. Black arrows indicate magnification and rotation to view the methionine pairs. (Top right) The methionine pair M1009-M391 near the entrance into the periplasmic cleft. (Bottom left) The methionine pair M486-M403 positioned in the middle of the transmembrane domain. (Bottom right) The methionine pair M501-M410 with side chains oriented away from each other near the entryway into the transmembrane domain from the cytosol.

Conformational change of a CusA monomer. (A) The final frame of a monomeric CusA 500-ns simulation starting from the “binding” state (red). This is structurally aligned with cryo-EM structures of the “extrusion” state (yellow) and “binding” state (blue). The spheres show the positions of the Cα atoms of N651 and R705. (B) Distribution of distances between Cα atoms of R705 and N651 over the last 100 ns of 500-ns simulations from the “extrusion” state with Cu-bound (E-Cu [panel III]), no Cu (E-Apo [panel IV]), “binding” state without Cu(I) (B-Apo [panel II]) and “binding” state with Cu(I) (B-Cu [panel I]). (C) The final frame of a monomeric CusA 500-ns simulation starting from an “extrusion” state with a Cu(I) ion docked into the binding site. This is structurally aligned with cryo-EM structures of the “extrusion” state (yellow) and “binding” state (blue). The spheres show the positions of the Cα atoms of N651 and R705.

Proton permeation path through the CusA protein. (A) An analysis of the pore profile along CusA (blue) with protonatable residues highlighted in red for acidic residues (D405 and E939) and blue for basic residues (K482 and K984). Pore-lining residues are highlighted in green for polar residues and brown for hydrophobic residues. Different subdomains are colored in purple (PC2), blue (PC1), orange (DC), yellow (DN), green (PN1), and red (PN2). (B) A path of proton permeation across the membrane via a water wire from basic residues (blue) to acidic residues (yellow) based on the “extrusion” state of a protomer from the EEE trimer. The structure displays a solvated pore after 20-ns equilibration with Cα restrained. Putative hydrogen bonds are shown as black dashes with distances in angstroms. (C) Calculated pK_a values of the four acidic and basic residues in the transmembrane region of the protein from the last 100 ns of the 450-ns simulation in the trimeric CusA pump with and without Cu(I). Data are shown for three repeats of the three subunits. Error bars display the standard errors of the means (n = 9). (D) Calculated number of water molecules involved in the water wire from the last 100 ns of the 450-ns simulation of trimeric CusA with (blue) and without (yellow) Cu(I). Data are shown for three repeats of the three subunits only in the subunit when water molecules are present in the pore. Error bars display the standard errors of the means (n = 8).