CopZ from Bacillus subtilis interacts in vivo with a copper exporting CPx-type ATPase CopA

Proc. Natl. Acad. Sci. U.S.A.

et al. 2006

146

252

Human Wilson protein is a copper-transporting ATPase located in the secretory pathway possessing six N-terminal metal-binding domains. Here we focus on the function of the metal-binding domains closest to the vesicular portion of the copper pump, i.e., domain 4 (WLN4), and a construct of domains 5 and 6 (WLN5-6). For comparison purposes, some experiments were also performed with domain 2 (WLN2). The solution structure of apoWLN5-6 consists of two ferredoxin folds connected by a short linker, and 15 N relaxation rate measurements show that it behaves as a unit in solution. An NMR titration of apoWLN5-6 with the metallochaperone Cu(I)HAH1 reveals no complex formation and no copper exchange between the two proteins, whereas titration of Cu(I)HAH1 with WLN4 shows the formation of an adduct that is in fast exchange on the NMR time scale with the isolated protein species as confirmed by 15 N relaxation data. A similar interaction is also observed between Cu(I)HAH1 and WLN2; however, the relative amount of the adduct in the protein mixture is lower. An NMR titration of apoWLN5-6 with Cu(I)WLN4 shows copper transfer, first to WLN6 then to WLN5, without the formation of an adduct. Therefore, we suggest that WLN4 and WLN2 are two acceptors of Cu(I) from HAH1, which then somehow route copper to WLN5-6, before the ATP-driven transport of copper across the vesicular membrane.ATPase function ͉ copper transport ͉ metal-binding domain ͉ metallochaperone ͉ Wilson disease protein

Section: Discussionmentioning

confidence: 99%

Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAH1 in copper uptake

Achila

Proc. Natl. Acad. Sci. U.S.A.

et al. 2006

146

252

“…Therefore, considering that a functional relation between CopA and CopZ from B. subtilis has been recently detected in vivo (27) and protein-protein interaction has been characterized in vitro (29), it is reasonable to conclude that the protein partner CopZ can release copper(I) to both metal binding sites independently. The partner protein-protein interaction was shown to occur between the negatively charged surface of CopZ and the positive surface of the second domain of CopA (29).…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, cytoplasmic copper chaperones such as Hah1 in humans, Atx1 in Saccharomyces cerevisiae, and CopZ in Bacillus subtilis and Enterococcus hirae bacteria, which deliver copper(I) to ATPases, share the same MXCXXC metal binding motif and the same ␤␣␤␤␣␤-fold (21)(22)(23)(24). In addition, interaction between the N-terminal region of the copper ATPase Ccc2 from S. cerevisiae and Atx1 as well as of the bacterial homologues, the copper ATPase CopA and the chaperone CopZ from B. subtilis, was demonstrated by yeast and bacterial two-hybrid assays, respectively (25)(26)(27). Complex formation of either Atx1 or CopZ with one N-terminal domain of either Ccc2 or CopA was also confirmed and characterized by in vitro NMR studies (28,29).…”

mentioning

confidence: 99%

Structural Basis for the Function of the N-terminal Domain of the ATPase CopA from Bacillus subtilis

Journal of Biological Chemistry

Ciofi‐Baffoni

et al. 2003

The solution structure of the N-terminal region (151 amino acids) of a copper ATPase, CopA, from Bacillus subtilis, is reported here. It consists of two domains, CopAa and CopAb, linked by two amino acids. It is found that the two domains, which had already been separately characterized, interact one to the other through a hydrogen bond network and a few hydrophobic interactions, forming a single rigid body. The two metal binding sites are far from one another, and the short link between the domains prevents them from interacting. This and the surface electrostatic potential suggest that each domain receives copper from the copper chaperone, CopZ, independently and transfers it to the membrane binding site of CopA. The affinity constants of silver(I) and copper(I) are similar for the two sites as monitored by NMR. Because the present construct "domain-short link-domain" is shared also by the last two domains of the eukaryotic copper ATPases and several residues at the interface between the two domains are conserved, the conclusions of the present study have general validity for the understanding of the function of copper ATPases.

“…In the CopZ͞Cu(I)͞CopAb complex (CopAb being the second Nterminal domain of the CopA ATPase from Bacillus subtilis), loop 1 of CopZ takes the conformation of the apo form rather than that of the copper form (7), which indicates that copper is released from the metallochaperone to the ATPase. ⌬copZ mutants were subsequently shown to be copper-sensitive (8), consistent with a role in export rather than import. It is anticipated that any analogous mechanism for ScAtx1 has somehow been adapted to force copper transfer to the metallochaperone from one P 1 -type ATPase and release to another, and His-61 has been suggested to play a significant role to drive the two distinct copper-transfer mechanisms (1).…”

mentioning

confidence: 94%

“…In common with related orthologues from other bacteria, yeast, and human (7)(8)(9)(10), ScAtx1 directly interacts in two hybrid assays (5) and in vitro studies (11) with the soluble, N-terminal domains of P 1 -type copper ATPases. However, unlike eukaryotic copper metallochaperones, which interact with a single ATPase, ScAtx1 associates with two such ATPases, PacS and CtaA.…”

mentioning

confidence: 98%

The delivery of copper for thylakoid import observed by NMR

Proc. Natl. Acad. Sci. U.S.A.

Ciofi‐Baffoni

et al. 2006

The thylakoid compartments of plant chloroplasts are a vital destination for copper. Copper is needed to form holo-plastocyanin, which must shuttle electrons between photosystems to convert light into biologically useful chemical energy. Copper can bind tightly to proteins, so it has been hypothesized that copper partitions onto ligand-exchange pathways to reach intracellular locations without inflicting damage en route. The copper metallochaperone Atx1 of chloroplast-related cyanobacteria ( PacS ͉ protein-protein interaction ͉ ScAtx1 ͉ metallochaperone P-type ATPase