Mechanistic insights into Cu(I) cluster transfer between the chaperone CopZ and its cognate Cu(I)-transporting P-type ATPase, CopA

Abstract

Multinuclear Cu(I) clusters are common in nature, but little is known about their formation or transfer between proteins. CopZ and CopA from Bacillus subtilis, which are involved in a copper-efflux pathway, both readily accommodate multinuclear Cu(I) clusters. Using the luminescence properties of a multinuclear Cu(I)-bound form of the two N-terminal soluble domains of CopA (CopAab) we have investigated the thermodynamic and kinetic properties of cluster formation and loss. We demonstrate that Cu(I)-bound forms of dimeric CopZ containing more than one Cu(I) per CopZ monomer can transfer Cu(I) to apo-CopAab, leading to the formation of luminescent dimeric CopAab. Kinetic studies demonstrated that transfer is a first-order process and that the rate-determining steps for transfer from CopZ to CopAab and vice versa are different processes. The rate of formation of luminescent CopAab via transfer of Cu(I) from CopZ was more rapid than that observed when Cu(I) was added 'directly' from solution or in complex with a cysteine variant of CopZ, indicating that transfer occurs via a transient protein-protein complex. Such a complex would probably require the interaction of at least one domain of CopAab with the CopZ dimer. Insight into how such a complex might form is provided by the high resolution crystal structure of Cu3(CopZ)3, a thus far unique trimeric form of CopZ containing a trinuclear Cu(I) cluster. Modelling studies showed that one of the CopZ monomers can be substituted for either domain of CopAab, resulting in a heterotrimer, thus providing a model for a 'trapped' copper exchange complex.

The Architecture of CopA from Archeaoglobus fulgidus Studied by Cryo-Electron Microscopy and Computational Docking

“…This conclusion applies both to the human pumps (ATP7A and ATP7B), which carry six MBD’s on their N-terminus, and CopA from bacteria, which generally have a single N-terminal MBD. In particular, several studies have shown Cu transfer from metallochaperones to the MBD’s (Achila et al, 2006; Banci et al, 2005a; Banci et al, 2005b; Gonzalez-Guerrero and Arguello, 2008; Larin et al, 1999; Ralle et al, 2004; Singleton et al, 2009; Strausak et al, 2003) and from metallochaperones to the transport sites (Gonzalez-Guerrero et al, 2009). However, disruption of the MBDs by either truncation or mutation does not affect transport activity of bacterial pumps (Bal et al, 2001; Fan and Rosen, 2002; Mana-Capelli et al, 2003; Mandal and Arguello, 2003; Mitra and Sharma, 2001; Rice et al, 2006) and disruption of MBD on the human pumps mainly affect enzyme activation and trafficking (Cater et al, 2007; Huster and Lutsenko, 2003; Strausak et al, 1999; Tsivkovskii et al, 2001; Veldhuis et al, 2009b; Walker et al, 2002).…”

Direct and Indirect Regulation of the ycnKJI Operon Involved in Copper Uptake through Two Transcriptional Repressors, YcnK and CsoR, in Bacillus subtilis

“…Thus, we speculate that the C-terminal region of the YcnK subunit encompasses one copper ion (probably monovalent) with a structural arrangement analogous to that of NosL, but the mode of coordination to copper ion is different in YcnK and NosL. Nevertheless, we also assume that Cys residue(s) play critical roles in the copper ion binding of YcnK, as observed in many metal ion binding proteins, including NosL, CsoR, and CopZ (11,20,22,31,46). According to our ortholog clustering search, the ycnKJI operon is conserved in a narrow subgroup of the Bacillus genus, comprising B. subtilis, B In general, the DeoR family members bind to several operator sites with widely varying configurations within the regulatory regions of the target genes.…”

“…In a previous study, it was reported that strong induction of the copZA operon occurs in the presence of copper ion, although it is not induced by the other metal ions tested, and that the CopZA system is required for resistance to a high level of copper ion (13), which accentuates its specialized function in copper export. The CopA protein, belonging to the integral membrane protein family of P-type ATPases, functions to translocate Cu ϩ across the cytoplasmic membrane, while CopZ is an Atx1-like soluble protein playing a role in the Cu ϩ transfer to the N-terminal domain of the CopA protein; this domain shows structural similarity to CopZ and transiently interacts with it when the transfer occurs (4,30,31). It seems that CopZ also serves as a copper chaperone with high binding affinity, so that the reactive free copper ion is kept at a very low level in the B. subtilis cell, as demonstrated for the yeast cell (26) (Fig.…”

Metallochaperones - an Overview

“…CopZ and CopA from Bacillus subtilis involved in a copper-efflux pathway readily accommodated multinuclear Cu(I) clusters.. Scientists have demonstrated that Cu(I)-bound forms of dimeric CopZ containing more than 1 Cu(I) per CopZ monomer can transfer Cu(I) to apo-CopAab leading to the formation of luminescent dimeric CopAab and the rate of formation of luminescent CopAab via transfer of Cu(I) from CopZ was more rapid than that observed when Cu(I) was added 'directly' from solution or in complex with a Cys variant of CopZ, indicating, thereby, that transfer occurred via a transient protein-protein complex [29]. CopZ and CopA from Bacillus subtilis involved in a copper-efflux pathway readily accommodated multinuclear Cu(I) clusters.. Scientists have demonstrated that Cu(I)-bound forms of dimeric CopZ containing more than 1 Cu(I) per CopZ monomer can transfer Cu(I) to apo-CopAab leading to the formation of luminescent dimeric CopAab and the rate of formation of luminescent CopAab via transfer of Cu(I) from CopZ was more rapid than that observed when Cu(I) was added 'directly' from solution or in complex with a Cys variant of CopZ, indicating, thereby, that transfer occurred via a transient protein-protein complex [29].…”