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

Abstract

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

Conserved residues modulate copper release in human copper chaperone Atox1

“…1A). Structural work has demonstrated that Cu chaperones and target metal-binding domains from various organisms possess the same fold and coordinate the metal via two surfaceexposed cysteines in the MXCXXC motif (13)(14)(15)(16)(17)(18)(19)(20)(21). Although the methionine in the first position of the MXCXXC motif is conserved in all organisms, it is not directly involved in metal ligation (13).…”

Mechanism of Cu+-transporting ATPases: Soluble Cu+chaperones directly transfer Cu+to transmembrane transport sites

“…In addition, CopZ has an unusual 130-aa Cys-rich N-terminal domain (N-CopZ) containing a [2Fe-2S] cluster and a Zn 2ϩ -binding site. The transfer of Cu ϩ from Cu ϩ chaperones to Cu ϩ -ATPase N-MBDs has been extensively characterized (9,14,18,27,28,32,33). Most of these studies have built on a model where a thermodynamically shallow gradient allows for Cu ϩ routing from the chaperone to MBDs and from these to the TM-MBS (represented by dashed red lines in Fig.…”

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

“…They were based on electron cryomicroscopy structures, into which related, known structures were fitted (Chintalapati et al 2008). Partial structures are available for isolated, soluble domains of CPx-type ATPases: several structures have been published for the unique N-terminal metalbinding domains of copper ATPases [heavy metal associated (HMA) domains (Achila et al 2006;Banci et al 2002Banci et al , 2000]. The HMA-domains appear to be modular structures which are present in one to six copies at the N terminus of CPx-type ATPases.…”