Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

Jensen, Pia Y.; Bonander, Nicklas; Møller, Lisbeth Birk; Farver, Ole

doi:10.1016/s0167-4838(99)00161-2

Cited by 34 publications

(20 citation statements)

References 35 publications

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“…With the exception of WD14, the K a values for the Wilson domains are all on the order of 10 5 to 10 6 M Ϫ1 . Affinities of this magnitude have been reported previously for the six MNK domains (43) and are consistent with EC 50 values measured for the activation of WND by copper (28,44). Importantly, the K a value for WD16, the most physiologically relevant variant, is essentially the same as that for Atox1 (Table II).…”

Section: Discussionsupporting

confidence: 89%

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

Wernimont¹,

Yatsunyk

Rosenzweig

2004

Journal of Biological Chemistry

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The Wilson disease protein (WND) is a transport ATPase involved in copper delivery to the secretory pathway. Mutations in WND and its homolog, the Menkes protein, lead to genetic disorders of copper metabolism. The WND and Menkes proteins are distinguished from other P-type ATPases by the presence of six soluble N-terminal metal-binding domains containing a conserved CXXC metal-binding motif. The exact roles of these domains are not well established, but possible functions include exchanging copper with the metallochaperone Atox1 and mediating copper-responsive cellular relocalization. Although all six domains can bind copper, genetic and biochemical studies indicate that the domains are not functionally equivalent. One way the domains could be tuned to perform different functions is by having different affinities for Cu(I). We have used isothermal titration calorimetry to measure the association constant (K a ) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to their metallochaperone Atox1. The association constants for both the chaperone and target domains are ϳ10 5 to 10 6 M ؊1 , suggesting that the handling of copper by Atox1 and copper transfer between Atox1 and WND are under kinetic rather than thermodynamic control. Although some differences in both n and K a values are observed for variant proteins containing less than the full complement of six metal-binding domains, the data for domains 1-6 were best fitted with a single site model. Thus, the individual functions of the six WND metal-binding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties.

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Section: Discussionsupporting

confidence: 89%

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

Wernimont¹,

Yatsunyk

Rosenzweig

2004

Journal of Biological Chemistry

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“…2C (3,26,35). However, lower K a values have been obtained in studies of similar domains by using alternative methodologies (24,36). Consequently, the emphasis here will be placed on the comparative analysis of Cu ϩ -binding parameters of CopZ and CopA domains obtained with the same method.…”

Section: Resultsmentioning

confidence: 99%

“…They have a ␤␣␤␤␣␤ fold and an invariant CXXC metal-binding sequence similar to the well described Cu ϩ chaperones, Atox1, Atx1, and CopZ (20,22,23). N-MBDs bind Cu ϩ with high affinity (24)(25)(26), and in vivo, they receive Cu ϩ from the corresponding Cu ϩ chaperones (23,27,28). In bacterial Cu ϩ -ATPases, deletion of these domains or mutation of Cu ϩ -binding Cys residues does not prevent metal activation of the ATPase, although they affect enzyme turnover rate (15,16,19).…”

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confidence: 99%

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

González‐Guerrero

Argüello

2008

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

219

239

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As in other P-type ATPases, metal binding to transmembrane metal-binding sites (TM-MBS) in Cu ؉ -ATPases is required for enzyme phosphorylation and subsequent transport. However, Cu ؉ does not access Cu ؉ -ATPases in a free (hydrated) form but is bound to a chaperone protein. CopA ͉ CopZ ͉ Cu homeostasis ͉ Cu-ATPase ͉ metal binding C opper is an essential cofactor in many biological processes (1). However, it also participates in harmful Fenton reactions. Consequently, Cu is ''buffered'' at a ''no-free Cu'' level by metallothioneins and chaperones with binding constants for Cu ϩ in the picomolar-femtomolar range (2, 3). Within these constraints, Cu ϩ chaperones route Cu ϩ to various intracellular targets, and Cu ϩ transmembrane transport systems maintain the total copper quota within the 10-100 M range (1-4). How the Cu ϩ chaperones transfer the metal to and from transmembrane transport sites is a central feature of transmembrane Cu ϩ transport. To better understand these phenomena, we have studied the delivery of Cu ϩ by the Archaeoglobus fulgidus Cu ϩ chaperone, CopZ, to the corresponding Cu ϩ -ATPase, CopA.CopA is a member of the P 1B subgroup of P-type ATPases (5-7). Cu ϩ -ATPases are essential to maintain Cu ϩ homeostasis. For instance, mutations in the two Cu ϩ -ATPase genes present in humans, ATP7A and ATP7B, lead to Menkes syndrome and Wilson's disease, respectively (8, 9). The Cu ϩ -ATPases transport cycle follows the classical E1/E2 Albers-Post model (10-12). Catalytic phosphorylation of the enzyme in the E1 conformation occurs upon binding of cytoplasmic metal to transmembrane metal-binding sites (TM-MBS) and ATP binding with high affinity (l M) to the ATP-binding domain (ATP-BD) (Fig. 1). It is assumed that upon phosphorylation, Cu ϩ is occluded within the transmembrane region. The subsequent conformational change allows metal deocclusion and release to the extracellular (vesicular/luminal) compartment followed by enzyme dephosphorylation and return to the E1 form (10). Functional studies of various Cu ϩ -ATPases have characterized the Cu ϩ transport, Cu ϩ -dependent ATPase activity, phosphorylation, and dephosphorylation partial reactions (5,(13)(14)(15)(16)(17)(18)(19).Cu ϩ -ATPases consist of eight transmembrane segments, two large cytosolic loops comprising the A-domain and the ATP-BD, and regulatory metal-binding domains (MBDs) in their N terminus (6, 8-10, 20, 21) (Fig. 1). A. fulgidus CopA has an atypical

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“…The work from several laboratories yielded information about copper binding properties of MBS (5,24,25) and their relative importance for overall function of MNKP and WNDP (11,12). However, specific functions of the N-terminal MBS are yet to be understood.…”

Section: Discussionmentioning

confidence: 99%

The Distinct Roles of the N-terminal Copper-binding Sites in Regulation of Catalytic Activity of the Wilson's Disease Protein

Hüster¹,

Lutsenko

2003

Journal of Biological Chemistry

107

125

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Wilson's disease protein (WNDP) is a copper-transporting ATPase essential for normal distribution of copper in human cells. Recent studies demonstrate that copper regulates WNDP through several mechanisms. Six metal-binding sites (MBS) at the N terminus of WNDP are predicted to be involved in copper-dependent regulation of WNDP; however, specific roles of MBS remain poorly understood. To address this issue, we generated WNDP variants with mutations or truncation in the N-terminal region and characterized their functional properties. We show that copper cooperatively stimulates catalytic activity of WNDP and that this effect requires the presence of both MBS5 and MBS6. Mutations of MBS6 or MBS1-5 result in non-cooperative activation of the enzyme by copper, whereas the deletion of MBS1-4 does not abolish cooperativity. Our data further suggest that MBS5 and MBS6 together regulate the affinity of the intramembrane-binding site(s) for copper. Analysis of the copper-dependent stimulation of catalytic phosphorylation demonstrate that the MBS6 and MBS1-5 mutants have a 7-8-fold lower EC 50 for copper activation, suggesting that their affinity for copper is increased. This conclusion is confirmed by a markedly decreased inhibition of these mutants by a copper chelator bathocuproine disulphonate. In contrast, deletion of MBS1-4 does not affect the affinity of sites important for catalytic phosphorylation. Rather, the MBS1-4 region appears to control access of copper to the functionally important metal-binding sites. The implications of these findings for intracellular regulation of WNDP are discussed. Wilson's disease protein (WNDP)1 is a copper-transporting P 1 -type ATPase essential for regulation of copper concentration in human cells. The major functional activity of WNDP is to couple the energy of ATP hydrolysis with transport of copper from cytosol across cell membranes (1, 2). During ATP hydrolysis, WNDP becomes transiently phosphorylated at the invariant residue Asp-1027; catalytic phosphorylation from ATP is specifically stimulated by copper (3). Following copper translocation across the cell membrane, WNDP is dephosphorylated. In vitro, catalytic phosphorylation can also be achieved using inorganic phosphate (P i ) in the presence of Mg 2ϩ ; this reaction reflects the reversibility of the dephosphorylation step (4). Similarly to other P-type ATPases, WNDP is thought to cycle between two major conformations: E1, in which WNDP binds copper and ATP with high affinity and becomes phosphorylated, and E2, in which the affinity for copper and ATP is low but WNDP can still be phosphorylated using inorganic phosphate.WNDP has multiple copper-binding sites located at two different regions of the protein (Fig. 1A). In the N-terminal portion of WNDP, there are six metal-binding sites (MBS) formed by cysteine residues of the sequence motifs GMXCXXC. Each motif belongs to a repetitive 70 amino acid sequence and binds one copper in the reduced Cu ϩ1 form (5, 6). In addition to six cytosolic copper-binding sites, there is at ...

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Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

Cited by 34 publications

References 35 publications

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

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

The Distinct Roles of the N-terminal Copper-binding Sites in Regulation of Catalytic Activity of the Wilson's Disease Protein

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Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

Cited by 34 publications

References 35 publications

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

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

The Distinct Roles of the N-terminal Copper-binding Sites in Regulation of Catalytic Activity of the Wilson's Disease Protein

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