Molecular Basis of the Cooperative Binding of Cu(I) and Cu(II) to the CopK Protein from Cupriavidus metallidurans CH34

Abstract

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 Cu(I) binding site generates a high-affinity Cu(II) binding site, and the high-affinity Cu(II) binding enhances Cu(I) binding. Native CopK and targeted variants were examined by chromatographic, spectroscopic, and X-ray crystallographic probes. Structures of two distinct forms of Cu(I)Cu(II)-CopK were defined, and structural changes associated with occupation of the Cu(II) site were demonstrated. In solution, monomeric Cu(I)Cu(II)-CopK features the previously elucidated Cu(I) site in Cu(I)-CopK, formed from four S(δ) atoms of Met28, -38, -44, and -54 (site 4S). Binding of Cu(I) to apo-CopK induces a conformational change that releases the C-terminal β-strand from the β-sandwich structure. In turn, this allows His70 and N-terminal residues to form a large loop that includes the Cu(II) binding site. In crystals, a polymeric form of Cu(I)Cu(II)-CopK displays a Cu(I) site defined by the S(δ) atoms of Met26, -38, and -54 (site 3S) and an exogenous ligand (modeled as H(2)O) and a Cu(II) site that bridges dimeric CopK molecules. The 3S Cu(I) 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 Cu(I) transfer processes. The adaptable nature of the Cu(I) 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.

The functional roles of the three copper sites associated with the methionine-rich insert in the multicopper oxidase CueO fromE. coli

“…Support for this requirement is provided by the observation that another bacterial periplasmic protein CopK binds Cu(I) with affinity indistinguishable to that of PcoC, 15 but the air-stable complex Cu I Cu II -CopK is not a Cu(I) substrate for CueO in 3-(N-morpholino)propanesulfonate (Mops) buffer whereas the air-stable Cu I Cu II -PcoC is oxidised rapidly under the same conditions. 9 The Cu(I) atom in CopK is bound close to an hydrophobic core [15][16][17] and appears to be protected against direct molecular contact with CueO. On the other hand, the Cu(I) site in PcoC lies on a flexible Met-rich metal binding loop exposed to the protein surface 14,18 and is likely to interact directly with CueO for efficient Cu(I) transfer and oxidation.…”

Metal Response in Cupriavidus metallidurans

“…The NMR solution structure of Cu + Cu 2+ -CopK is constituted of two short antiparallel β-sheets and a turn of 3:10 helix (Ash et al 2011). As already observed in the structure of Cu + -CopK, the C-terminal strand is lost.…”

“…As the 3S and 4S sites share two Cu + ligands, M38 and M54, the two sites can be occupied one at a time. Experimental evidence has come from the study of a M44L mutant showing that the Cu + ion was locked at site 3S, and that Cu + -M44L-CopK remained in the dimeric state and was unable to induce the major change in protein conformation required for Cu 2+ binding (Ash et al 2011).…”

“…As already observed in the structure of Cu + -CopK, the C-terminal strand is lost. The Cu + ion occupies the solvent-inaccessible 4S site while the Cu 2+ ion is located in unstructured regions corresponding to residues 1-6 and 62-74 (Ash et al 2011). Absence of amide resonance of H70 in the 1 H, 15 N-HSQC spectrum of native Cu + Cu 2+ -CopK suggested that this residue contributes to Cu 2+ binding (Ash et al 2011).…”

“…The Cu + ion occupies the solvent-inaccessible 4S site while the Cu 2+ ion is located in unstructured regions corresponding to residues 1-6 and 62-74 (Ash et al 2011). Absence of amide resonance of H70 in the 1 H, 15 N-HSQC spectrum of native Cu + Cu 2+ -CopK suggested that this residue contributes to Cu 2+ binding (Ash et al 2011). Substitution of H70 by a phenylalanine residue showed that Cu + -H70F-CopK is not able to bind Cu 2+ , demonstrating that this histidine residue is involved in the Cu 2+ coordination site (Chong et al 2009;Ash et al 2011).…”