文摘
The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions. Upon copper challenge, it upregulates the periplasmic protein CopK (8.3 kDa). The function of CopK in the copper resistance response is ill-defined, but CopK demonstrates an intriguing cooperativity: occupation of a high-affinity CuI binding site generates a high-affinity CuII binding site, and the high-affinity CuII binding enhances CuI binding. Native CopK and targeted variants were examined by chromatographic, spectroscopic, and X-ray crystallographic probes. Structures of two distinct forms of CuICuII-CopK were defined, and structural changes associated with occupation of the CuII site were demonstrated. In solution, monomeric CuICuII-CopK features the previously elucidated CuI site in CuI-CopK, formed from four S未 atoms of Met28, -38, -44, and -54 (site 4S). Binding of CuI to apo-CopK induces a conformational change that releases the C-terminal 尾-strand from the 尾-sandwich structure. In turn, this allows His70 and N-terminal residues to form a large loop that includes the CuII binding site. In crystals, a polymeric form of CuICuII-CopK displays a CuI site defined by the S未 atoms of Met26, -38, and -54 (site 3S) and an exogenous ligand (modeled as H2O) and a CuII site that bridges dimeric CopK molecules. The 3S CuI binding mode observed in crystals was demonstrated in solution in protein variant M44L where site 4S is disabled. The intriguing copper binding chemistry of CopK provides molecular insight into CuI transfer processes. The adaptable nature of the CuI coordination sphere in methionine-rich clusters allows copper to be relayed between clusters during transport across membranes in molecular pumps such as CusA and Ctr1.