Mechanism of ATPase-mediated Cu+ export and delivery to periplasmic chaperones: the interaction of Escherichia coli CopA and CusF

Abstract

Cellular copper homeostasis requires transmembrane transport and compartmental trafficking while maintaining the cell essentially free of uncomplexed Cu(2+/+). In bacteria, soluble cytoplasmic and periplasmic chaperones bind and deliver Cu(+) to target transporters or metalloenzymes. Transmembrane Cu(+)-ATPases couple the hydrolysis of ATP to the efflux of cytoplasmic Cu(+). Cytosolic Cu(+) chaperones (CopZ) interact with a structural platform in Cu(+)-ATPases (CopA) and deliver copper into the ion permeation path. CusF is a periplasmic Cu(+) chaperone that supplies Cu(+) to the CusCBA system for efflux to the extracellular milieu. In this report, using Escherichia coli CopA and CusF, direct Cu(+) transfer from the ATPase to the periplasmic chaperone was observed. This required the specific interaction of the Cu(+)-bound form of CopA with apo-CusF for subsequent metal transfer upon ATP hydrolysis. As expected, the reverse Cu(+) transfer from CusF to CopA was not observed. Mutation of CopA extracellular loops or the electropositive surface of CusF led to a decrease in Cu(+) transfer efficiency. On the other hand, mutation of Met and Glu residues proposed to be part of the metal exit site in the ATPase yielded enzymes with lower turnover rates, although Cu(+) transfer was minimally affected. These results show how soluble chaperones obtain Cu(+) from transmembrane transporters. Furthermore, by explaining the movement of Cu(+) from the cytoplasmic pool to the extracellular milieu, these data support a mechanism by which cytoplasmic Cu(+) can be precisely directed to periplasmic targets via specific transporter-chaperone interactions.

Keywords: Chaperone; Copper; Copper ATPase; Copper Transport; Metal Homeostasis; Metal Ion-Protein Interaction.

FIGURE 1.

Proposed Cu⁺-ATPase catalytic cycle. CopZ, the various cytoplasmic chaperones; CusF, the likely array of, depending on the organism, periplasmic, vesicular, or lumenal Cu⁺ chaperones.

FIGURE 2.

In silico analysis of EcCopA-EcCusF interaction. A, model of the EcΔN-CopA (gray)-EcCusF (blue) interaction generated by ClusPro. EcΔN-CopA was modeled using LpCopA (Protein Data Bank entry 3RFU) as template. Apo-EcCusF (Protein Data Bank entry 1ZEQ) is shown. Periplasmic loops (PL; green), putative exit residues Met²⁰⁴ and Glu²⁸⁷ (red) in EcΔN-CopA, and amino acids His³⁶, Met⁴⁷, and Met⁴⁹ forming the metal binding site in EcCusF (orange) are highlighted. B, zoom in to the interaction between the periplasmic loops of EcΔN-CopA (green) and EcCusF. C, sequences of EcCopA periplasmic loops.

FIGURE 3.

EcΔN-CopA Cu⁺ transfer to EcCusF. Cu⁺ transfer was initiated by the addition of ATP to EcΔN-CopA/EcCusF mixes in 1:1 (A), 1:2 (B), and 1:10 (C) molar ratios. The levels of Cu⁺ (red) and protein (gray) in the wash and elution fractions resulting from separating the proteins in Strep-tactin columns are shown. Values are the means ± S.E. (error bars) (n = 3). D, representative Western blots using anti-His₆ and anti-CusF antibodies of the peaks corresponding to EcΔN-CopA (W1) and EcCusF proteins (E2). E, percentage of Cu⁺ eluting with EcCusF (blue) and EcΔN-CopA (green).

FIGURE 4.

Specificity of EcΔN-CopA Cu⁺ transfer to EcCusF. A, Cu⁺ transfer from EcΔN-CopA to EcCusF upon the addition of ADP (red) and Cu⁺ transfer from EcΔN-CopA to EcCusF-M47I/M49I (green). B, Cu⁺ transfer from EcΔN-CopA to AfCt-CopZ (red) and Cu⁺ transfer from AfΔN,C-CopA to EcCusF (green). In all cases, ATPase·(Cu⁺)₂/chaperone were incubated in a 1:2 molar ratio. The Cu⁺ transfer from EcΔN-CopA to EcCusF in a 1:2 molar ratio (B) is included in blue for reference. C, percentage of Cu⁺ eluting with the chaperones (blue) and ATPases (green). Values are the means ± S.E. (error bars) (n = 3). Significant differences from the transfer observed from EcΔN-CopA to EcCusF (Fig. 3B) were determined by Student's t test. *, p ≤ 0.05.

FIGURE 5.

Conformational determinants of stable EcΔN-CopA3·EcCusF interactions. EcΔN-CopA and EcCusF were allowed to interact under indicated conditions and immunoprecipitated using an anti-EcCusF antibody. Representative Western blots (WB) of immunoprecipitated proteins using anti-His₆ (detecting EcΔN-CopA) and anti-EcCusF antibodies are shown.

FIGURE 6.

A, docking model of EcΔN-CopA with EcCusF highlighting mutations at the Cu⁺ binding site and electropositive surface. B, Cu⁺ transfer from EcΔN-CopA to EcCusF-5Ala. ATPase·(Cu⁺)₂/chaperone were incubated in a 1:2 molar ratio. The levels of Cu⁺ (red) and protein (gray) in the wash and elution fractions resulting from separating the proteins on Strep-tactin columns are shown. Cu⁺ transfer from EcΔN-CopA to EcCusF in a 1:2 molar ratio (Fig. 3B) is included in blue for reference. Values are the means ± S.E. (error bars) (n = 3). Significant differences in the transfer from EcΔN-CopA to EcCusF (Fig. 3B) were determined by Student's t test. *, p ≤ 0.05.

FIGURE 7.

Role of periplasmic loops in the EcΔN-CopA·EcCusF interactions. Shown are Cu⁺-dependent ATPase activity of EcΔN-CopA (blue line), EcΔN-CopA-M1L1 (red line), and EcΔN-CopA-M2L1 (red dotted line) (A); EcΔN-CopA (blue line), EcΔN-CopA-M2L2 (red line), and EcΔN-CopA-M1L2 (red dotted line) (B); and EcΔN-CopA (blue line) and EcΔN-CopA-M1L4 (red line) (C). Data points represent the mean ± S.E. (error bars) of at least three independent experiments performed in duplicate. ATPase activity curves were fit to ν = V_max [Cu⁺]/([Cu⁺] + K_½). Fitting parameters for all curves are presented in Table 1. D, Cu⁺ transfer from EcΔN-CopA M1L1, M2L1, M1L2, M2L2, and M1L4 to EcCusF was determined. Results are shown as the percentage of Cu⁺ eluting with the chaperones (blue) and ATPases (green). In all cases, ATPase·(Cu⁺)₂/chaperone were incubated in a 1:2 molar ratio. Values are the means ± S.E. (n = 3). Significant differences in the transfer from EcΔN-CopA to EcCusF (Fig. 3B) were determined by Student's t test; *, p ≤ 0.05.

FIGURE 8.

Role of EcΔN-CopA invariant Met²⁰⁴ and Glu²⁸⁷ in the Cu⁺ transport. A, Cu⁺-dependent ATPase activity of ΔN-CopA (blue line), M204A (red line), and M204C (red dotted line) mutants. B, E287A (red line) and E287C (red dotted line) mutants. Data points represent the mean ± S.E. (error bars) of at least three independent experiments performed in duplicate. ATPase activity curves were fit to ν = V_max [Cu⁺]/([Cu⁺] + K_½). Fitting parameters for all curves are presented in Table 1. C, Cu⁺ transfer from EcΔN-CopA M204A, M204C, E287A, and E287C to EcCusF was determined. Results are shown as the percentage of Cu⁺ eluting with the chaperones (blue) and ATPases (green). In all cases, ATPase·(Cu⁺)₂/chaperone were incubated in a 1:2 molar ratio. Values are the means ± S.E. (n = 3).