Unprecedented Binding Cooperativity between Cu^I and Cu^II in the Copper Resistance Protein CopK from Cupriavidus metallidurans CH34: Implications from Structural Studies by NMR Spectroscopy and X-Ray Crystallography

Abstract

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions, including copper. This ability arises primarily from the presence of a large plasmid pMOL30 which includes a cluster of 19 cop genes that respond to copper. One of the protein products CopK is induced at high levels and is expressed to the periplasm as a small soluble protein (8.3 kDa). Apo-CopK associates in solution to form a dimer (K(D) approximately 10(-5) M) whose structure was defined by NMR and X-ray crystallography. The individual molecules feature two antiparallel beta-sheets arranged in a sandwich-like structure and interact through C-terminal beta-strands. It binds Cu(II) with low affinity (K(D)(Cu(II)) > 10(-6) M) but Cu(I) with high affinity (K(D)(Cu(I)) = 2 x 10(-11) M). Cu(I)-CopK was also a dimer in the solid state and featured a distorted tetrahedral site Cu(I)(S-Met)(3)(NCS). The isothiocyanato ligand originated from the crystallization solution. Binding of Cu(I) or Ag(I), but not of Cu(II), favored the monomeric form in solution. While Ag(I)-CopK was stable as isolated, Cu(I)-CopK was moderately air-sensitive due to a strong binding cooperativity between Cu(I) and Cu(II). This was documented by determination of the Cu(I) and Cu(II) binding affinities in the presence of the other ion: K(D)(Cu(I)) = 2 x 10(-13) M and K(D)(Cu(II)) = 3 x 10(-12) M, that is, binding of Cu(II) increased the affinity for Cu(I) by a factor of approximately 10(2) and binding of Cu(I) increased the affinity for Cu(II) by a factor of at least 10(6). Stable forms of both Cu(I)Cu(II)-CopK and Ag(I)Cu(II)-CopK were isolated readily. Consistent with this unprecedented copper binding chemistry, NMR spectroscopy detected three distinct forms: apo-CopK, Cu(I)-CopK and Cu(I)Cu(II)-CopK that do not exchange on the NMR time scale. This information provides a valuable guide to the role of CopK in copper resistance.

The Lumenal Loop Met672–Pro707 of Copper-transporting ATPase ATP7A Binds Metals and Facilitates Copper Release from the Intramembrane Sites

“…For both copper binding stoichiometries, the Cu(I)-N(His) bond length was 1.88 Ϯ 0.2 Å, a value typical of linear 2-coordination in either Cu(I)N 2 (22, 23) or mixed Cu(I)NS environments (24). The Cu(I)-S distance was 2.23 Ϯ 0.2 Å, a value closer to the typical 3-coordinate distance of 2.25-2.30 Å (17,18,(25)(26)(27). Altogether, the EXAFS data confirmed the presence of at least two structurally non-equivalent Cu(I)-binding sites.…”

Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

“…The preference of Cu(I) for soft bases (His, Cys, and Met) is manifest in the coordination environments of cytoplasmic metallochaperones (35)(36)(37) and the Met-rich sites found in periplasmic copperbinding proteins (38)(39)(40)(41). However, in the context of Cu(I) transport through membrane channels, kinetic lability may be more important than thermodynamic stability, and indeed high binding affinity is likely to inhibit transport, unless accompanied by energy input that can toggle high-and low-affinity states via conformational change, as has been proposed as a mechanism for intramembrane transport in P1B-type ATPases (42).…”

The CopRS Two-Component System Is Responsible for Resistance to Copper in the Cyanobacterium Synechocystis sp. PCC 6803

“…CopM contained an elevated number of Met and His residues and a signal peptide that will target it to the periplasmic and/or thylakoid compartment. In other copper resistance systems, periplasmic proteins with an elevated number of these residues work as copper chaperones, acting either as a buffer and/or transferring periplasmic copper to RND transport systems (Loftin et al, 2005;Bagai et al, 2008;Chong et al, 2009;Mealman et al, 2011) that efflux it outside the cell. Attempts to delete copM without affecting copRS expression have been unsuccessful, and for that reason we could not determine the contribution of CopM to copper resistance.…”