Important Structural Features of Thiolate-Rich Four-Helix Bundles for Cu(I) Uptake and Removal

[Image: see text] A family of bacterial copper storage proteins (the Csps) possess thiolate-lined four-helix bundles whose cores can be filled with Cu(I) ions. The majority of Csps are cytosolic (Csp3s), and in vitro studies carried out to date indicate that the Csp3s from Methylosinus trichosporium OB3b (MtCsp3), Bacillus subtilis (BsCsp3), and Streptomyces lividans (SlCsp3) are alike. Bioinformatics have highlighted homologues with potentially different Cu(I)-binding properties from these characterized “classical” Csp3s. Determination herein of the crystal structure of the protein (RkCsp3) from the methanotroph Methylocystis sp. strain Rockwell with Cu(I) bound identifies this as the first studied example of a new subgroup of Csp3s. The most significant structural difference from classical Csp3s is the presence of only two Cu(I) sites at the mouth of the bundle via which Cu(I) ions enter and leave. This is due to the absence of three Cys residues and a His-containing motif, which allow classical Csp3s to bind five to six Cu(I) ions in this region. Regardless, RkCsp3 exhibits rapid Cu(I) binding and the fastest measured Cu(I) removal rate for a Csp3 when using high-affinity ligands as surrogate partners. New experiments on classical Csp3s demonstrate that their His-containing motif is not essential for fast Cu(I) uptake and removal. Other structural features that could be important for these functionally relevant in vitro properties are discussed.