Bacterial cytosolic proteins with a high capacity for Cu(I) that protect against copper toxicity

Abstract

Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis. These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1–2) × 1017 M−1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria.

Visualizing Biological Copper Storage: The Importance of Thiolate‐Coordinated Tetranuclear Clusters

“…Cu I ions populate numerous sites as the bundle fills, with a clear preference for those towards the center, and particularly in the Cu3–Cu6, Cu7–Cu10, and Cu11–Cu14 tetranuclear clusters (Figure S1 in the Supporting Information). This behavior is consistent with in vitro Cu I ‐binding properties,7 and a more ordered uptake mechanism for Mt Csp3 compared to Mt Csp1 (for further discussion see the Supporting Information). The total occupancies of the Cu3–Cu6, Cu7–Cu10, and Cu11–Cu14 clusters range from 2.4 to 2.7 in the 8 equiv structure and increase to 3.5 to 3.7 in 14 equiv Mt Csp3 (Figures 2, 3, and Figure S2 and Tables S2, S3 in the Supporting Information).…”

“…2 molar equiv of Cu I (see Supporting Information6, 7, 13, 14, 15) has four partially occupied sites (Figure 1 and Table S1 in the Supporting Information). These are Cu11 with an occupancy of 0.25, and Cu12, Cu13, and Cu14, all with occupancies of 0.35, which form a symmetrical tetranuclear cluster (Figure 1 b,c).…”

Bacterial copper storage proteins

“…Atypical coordination is again found at the mouth of the bundle where His110 binds Cu18 along with Cys111, and His104 ligates Cu19 in addition to two thiolates ( Fig. 3B) (36) ) is similar to that of MtCsp1 (35,36).…”

“…In vitro studies of MtCsp3 show it is also a tetramer of four-helix bundles having 18 Cys residues pointing into the core of each monomer (Fig. 3A) (36). The additional Cys residues, compared to MtCsp1, are found in CXXXC motifs, and the protein also has no disulfide bonds present.…”

“…In the case of MtCsp3 Cu(I) binding gives rise to relatively intense fluorescence at ~600 nm upon excitation within the S(Cys)→Cu(I) ligand-to-metal charge transfer bands below 400 nm (35,36). Such emission has been associated with Cu(I)-Cu(I) interactions in proteins binding solvent-protected Cys-coordinated Cu(I) clusters, such as the MTs (38,42,66).…”