A new structural paradigm in copper resistance in Streptococcus pneumoniae

Abstract

Copper resistance has emerged as an important virulence determinant of microbial pathogens. In Streptococcus pneumoniae, copper resistance is mediated by the copper-responsive repressor CopY, CupA and the copper-effluxing P(1B)-type ATPase CopA. We show here that CupA is a previously uncharacterized cell membrane-anchored Cu(I) chaperone and that a Cu(I) binding-competent, membrane-localized CupA is obligatory for copper resistance. The crystal structures of the soluble domain of CupA and the N-terminal metal-binding domain (MBD) of CopA (CopA(MBD)) reveal isostructural cupredoxin-like folds that each harbor a binuclear Cu(I) cluster unprecedented in bacterial copper trafficking. NMR studies reveal unidirectional Cu(I) transfer from the low-affinity site on the soluble domain of CupA to the high-affinity site of CopA(MBD). However, copper binding by CopA(MBD) is not essential for cellular copper resistance, consistent with a primary role of CupA in cytoplasmic Cu(I) sequestration and/or direct delivery to the transmembrane site of CopA for cellular efflux.

Figure 1. Copper sensitivity phenotypes of mutant S. pneumoniae D39 strains

ΔcupA and ΔcopA Spn strains are highly sensitive to copper toxicity (a) which can be reversed by expression of cupA from a heterologous promoter (b). (c) Deletion of the single putative transmembrane helix abrogates copper resistance to an extent comparable to inactivation of CopA(D442A) (compare to Supplementary Fig. 4d). In all cases, two independent isolates of the same strain designation were constructed and duplicate (or more) growth experiments were carried out with each of the two strains. (d) Both CopA and CupA localize to the cell membrane fraction. The results of a subcellular fractionation of copA-(C)-FLAG (IU6044) (top) and cupA-(C)-FLAG (IU6041) (bottom) with visualization by anti-FLAG western blotting. Supernatants (S) or pellets (P) are marked for centrifugation steps, and cell fractions are indicated below the blots. See Supplementary Figure 6 for additional experimental details and the full blot and Supplementary Tables 1 and 2 for strain details.

Figure 2. Crystallographic structures of sCupA and CopA^MBD

Structure representations of sCupA (a–c) and CopA^MBD (d–f). Close-up views of the Cu(I) coordination geometries of each protein are shown (b, e) as are solvent-accessible surface areas (arbitrarily colored according to residue type) (c, f) around each binuclear Cu(I) chelate. Electrostatic surface potentials (painted based on surface potentials) of sCupA (g) and CopA^MBD (h). Structure statistics compiled in Supplementary Table 3.

Figure 3. The Met-rich S2 site is the low-affinity site on both CopA^MBD and sCupA and Cu(I) is transferred only from the S2 site of sCupA to the S1 site of apo-MBD

X-ray absorption near-edge spectra (XANES) of Cu₁ CopA^MBD (a) and Cu₁ sCupA (b) in the presence of 0.2 M NaBr (red) or 0.2 M NaCl (blue). Overlay of the Met thioether methyl (¹³Cε-¹Hε) region of an ¹H,¹³C-HSQC spectrum for CopA^MBD (c) and sCupA (d) acquired in the apo-state (magenta), Cu₁ state (green) and Cu₂ states (blue). Cyan crosspeaks result when apo-MBD is mixed with 2.0 mol equiv of Cu₂ sCupA (c) or 1.0 mol equiv of Cu₂ sCupA (d). Overlay of the backbone ¹H,¹⁵N-HSQC spectra of CopA^MBD (e) and sCupA (f), with the same crosspeak color pattern as in panels c,d.

Figure 4. NMR chemical shift perturbation analysis of sCupA and CopA^MBD induced by Cu(I) binding

Ribbon representation of the changes in backbone amide chemical shift upon Cu(I) binding by sCupA (a,b) and CopA^MBD (c,d). Panels a and c represent Δppm (Cu₁–apo) while panels b and d represent Δppm (Cu₂–Cu₁). The ribbon is painted white for Pro residues and black for those resonances broadened beyond detection in the apo-state in each case.

Figure 5. Mutagenesis of CupA Cu(I) binding residues partly or completely abrogates Cu(I) resistance by S. pneumoniae, but not in CopA^MBD

Representative growth curves for the indicated S. pneumoniae strains in BHI in the absence (a) or presence (b,c) of 0.2 mM Cu(II) added to the growth medium. In all cases, two independent isolates of the same strain designation were constructed and duplicate (or more) growth experiments were carried out with each of the two strains. See Supplementary Tables 1 and 2 for strain details.