RESEARCH ARTICLE

Identification and Analysis of 21 NovelDisease-Causing Amino Acid Substitutionsin the Conserved Part of ATP7A

Lisbeth Birk M�ller,1* Jens Thostrup Bukrinsky,2–4 Anne M�lgaard,2,5 Marianne Paulsen,1 Connie Lund,1

Zeynep Tumer,6 Sine Larsen,2 and Nina Horn1

1Kennedy Institute-Statens Øjenklinik, Glostrup, Denmark; 2Centre for Crystallographic Studies, University of Copenhagen, Copenhagen,Denmark; 3Carlsberg Laboratories, Valby, Denmark; 4Novo Nordisk A/S, Denmark, Bagsværd, Denmark; 5Center for Biological SequenceAnalysis, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark; 6Wilhelm Johannsen Centre for Functional Genome Research,University of Copenhagen, Copenhagen, Denmark

Communicated by Peter Byers

ATP7A encodes a copper-translocating ATPase that belongs to the large family of P-type ATPases. Eightconserved regions define the core of the P-type ATPase superfamily. We report here the identification of 21novel missense mutations in the conserved part of ATP7A that encodes the residues p.V842-p.S1404. Using thecoordinates of X-ray crystal structures of the sarcoplasmic reticulum Ca2+-ATPase, as determined in thepresence and absence of Ca2+, we created structural homology models of ATP7A. By mapping the substitutedresidues onto the models, we found that these residues are more clustered three-dimensionally than expectedfrom the primary sequence. The location of the substituted residues in conserved regions supports thefunctional similarities between the two types of P-type ATPases. An immunofluorescence analysis of Menkesfibroblasts suggested that the localization of a large number of the mutated ATP7A protein variants was correct.In the absence of copper, they were located in perinuclear regions of the cells, just like the wild type. However,two of the mutated ATP7A variants showed only partly correct localization, and in five cultures no ATP7Aprotein could be detected. These findings suggest that although a disease-causing mutation may indicate afunctional significance of the affected residue, this is not always the case. Hum Mutat 26(2), 84–93, 2005.rr 2005 Wiley-Liss, Inc.

KEY WORDS: ATP7A; copper transport; missense mutations; P-type ATPases; structure; localization; stability

INTRODUCTION

Menkes disease (MD; MIM# 309400) is an X-linked, multi-systemic lethal disorder of copper metabolism that is characterizedby progressive neurodegeneration, connective tissue abnormalities,and abnormal hair [Danks et al., 1972]. In addition to the severeclassic form of MD that leads to death in early childhood, milderforms are observed in about 5–10% of patients, with survival intoadulthood [Horn and Tumer, 2002]. The disorder is caused bymutations in the ATP7A gene (MIM# 300011) encoding ATP7A,a copper-transporting ATPase (EC 3.6.1.36) [Chelly et al., 1993;Vulpe et al., 1993; Mercer et al., 1993]. ATP7A is organized in 23exons that encode 1500 amino acid residues [Tumer et al., 1995;Dierick et al., 1995].

About 110 mutations have been reported in ATP7A in affectedpatients [reviewed in Tumer et al., 1999] (see the Human GeneMutation Database at http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html), only 14 of which are missense mutations [Daset al., 1994; Kaler, 1998; Levinson et al., 1997; Tumer et al., 1997,1999; Ambrosini and Mercer, 1999; Ogawa et al., 1999; Hahnet al., 2001; Gu et al., 2001].

ATP7A has a dual role. It is responsible for the copper loadingof several copper-requiring enzymes, and for the ATP-driven effluxof copper from the cell [Camakaris et al., 1995; Voskoboinik et al.,

1998; Payne and Gitlin, 1998]. ATP7A is normally localized to thetrans-Golgi network (TGN) [Yamaguchi et al., 1996], although inresponse to elevated copper conditions it is translocated to theplasma membrane [Petris et al., 1996].

ATP7A and ATP7B (mutations in ATP7B result in Wilsondisease (WD; MIM# 277900)) belong to subfamily IB of asuperfamily of membrane proteins termed P-type ATPases, whichare responsible for the active transport of cations across biologicalmembranes [M�ller et al., 1996]. The superfamily of P-typeATPases also includes the mammalian Na+/K+- and H+/K+

pumps (subfamily 2C), as well as plasma membrane andsarcoplasmatic reticulum Ca2+ pumps (subfamily 2B) [Axelsenand Palmgren, 1998]. Based on the crystal structure of rabbit

Received 10 June 2004; accepted revised manuscript 25 February2005.

nCorrespondence to: Lisbeth BirkM�ller, Kennedy Institute-Statens�jenklinik, Gl. Landevej 7,2600 Glostrup, Denmark.E-mail: lbm@kennedy.dk

Grant sponsors: Danish Medical Research Council; Novo NordiskFoundation; Foundation of1870; Danish National Research Council.

DOI 10.1002/humu.20190Published online inWiley InterScience (www.interscience.wiley.com).

rr2005 WILEY-LISS, INC.

HUMAN MUTATION 26(2), 84^93,2005

sarcoplasmic reticulum Ca2+ ATPase isoform 1a (ATP2A1)[Toyoshima et al., 2000], it has been postulated that all the P-type ATPases contain three cytoplasmic domains: the activation(A) domain, the phosphorylation (P) domain, and the nucleotide-binding (N) domain. A characteristic feature of the P-typeATPases is the DKTGT motif in the P domain, which containsthe invariant aspartate residue that is phosphorylated during thereaction cycle [Lutsenko and Kaplan, 1996]. A multiple-sequencealignment, covering the full length of the different P-typeATPases, demonstrates low overall similarity. However, the coresof the protein sequences align with a minimal number of insertionsand deletions [Axelsen and Palmgren, 1998], which suggests thatthe proteins have a very similar fold.

Because of the large size and the transmembrane andmultidomain structure of ATP7A, as well as the instability ofthe cDNA sequence [La Fontaine et al., 1998], it is difficult toexpress and purify ATP7A, and therefore functional studies arelimited [Strausak et al., 1999; Voskoboinik et al., 1999]. Thefunctional effect of a few disease-causing mutations in the ATP7Agene have been determined in studies using a yeast complementa-tion assay based on the ability of ATP7A to complement thecopper-loading deficiency of the yeast mutant ccc2, which lacksthe yeast homologue of ATP7A/ATP7B [Payne and Gitlin, 1998;Voskoboinik et al., 2001]. Studies using Cop B of Enterococcushirae as a model system have also been performed [Bissig et al.,2001a]. Also, a system for synthesizing wild-type ATP7A and anATP7A-GFP fusion protein in Xenopus laevis oocytes waspublished [Bissig et al., 2001b].

Modeling offers the very first approximation to a possiblestructural arrangement in ATP7A. Therefore, a supplementaryway to investigate the role of specific residues in ATP7A is tostudy the location of amino acid substitutions that lead to MD inthe three-dimensional (3D) model of the protein. Here we reportthe identification of 21 novel missense mutations and theirstructural locations, and the structural location of previouslyidentified missense mutations located in the conserved part ofATP7A that comprises the residues p.V842–p.S1404. Weconstructed 3D models in the presence and absence of Cu+ ofthe conserved core region of ATP7A by homology modeling, basedon published crystal structures of ATP2A1 [Toyoshima et al.,2000; Toyoshima and Nomura, 2002]. Finally, we analyzed Menkesfibroblasts for the presence and localization of the differentmutated ATP7A protein variants to determine whether anexpected functional effect was supported by the expressionpattern.

MATERIALSANDMETHODSPatients

The clinical symptoms of the patients suggested classic MD or avariant form of MD, and they were referred to the John F. KennedyInstitute for biochemical and molecular confirmation of thediagnosis. The patients showed clinical heterogeneity, as demon-strated by the variation in the progression and severity of theneurological symptoms, and the age of death. Patients with themildest form, occipital horn syndrome (OHS), survive intoadulthood and have predominantly connective-tissue symptoms[Horn and Tumer, 2002]. Classification of the phenotype is basedmainly on the clinical symptoms and/or age of death (Table 1).

Detection of Mutations in ATP7A

Genomic DNA was isolated from cultured skin fibroblasts orblood [Grimberg et al., 1989]. Mutation screening of the ATPTAgene was performed by single-strand conformation polymorphism

as described previously [Tumer et al., 1997] or by dideoxyfingerprinting modified from the method of Sarkar et al. [1992]. Inbrief, dideoxy fingerprinting was performed by PCR amplificationof each exon with the previously described primers [Tumer et al.,1997]. The PCR amplification of genomic DNA was performed ina total volume of 25 ml containing 100 nM of each primer, 20 mMof dNTP, and 0.25U of Taq polymerase. A set of terminationproducts was generated by limited sequencing of the amplifiedexon with a single dideoxy terminator, 32P-labeled primer, andThermoSequenase (United States Biochemical). The terminationproducts were separated on a 6% nondenaturing mutationdetection enhancement gel (FMC Bioproducts). Exons thatshowed an abnormal band pattern were sequenced directlywithout further purification. The mutation numbering is basedon the cDNA sequence, and +1 corresponds to the A of the ATGtranslation initiation codon in the reference sequence L06133.1.

Alignment of ATPases and Building of HomologyModels of ATP7A

The sequence alignment of ATP7A and ATP2A1 (Fig. 1) isbased on work by Axelsen and Palmgren [1998] and in an iterativeprocess with the model-building manually refined to enable a 3Dstructural alignment of ATP7A to ATP2A1. The alignment wascombined with the coordinates from the structures of ATP2A1(Protein Data Bank entries 1eul [Toyoshima et al., 2000] and liwo[Toyoshima and Nomura, 2002]) with the programs Swiss-modeland Swiss-pdb viewer [Peitsch, 1996; Guex and Peitsch, 1997].Turbo-Frodo [Roussel and Cambillau, 1991] and O [Jones et al.,1991] were used as graphical display programs to visually validateand analyze the models, and to adjust the side-chain conforma-tions. The differences between ATP2A1 and ATP7A at the N-terminal, the N domain, and the C-terminal are large and had tobe accounted for in the models. Although it is impossible toachieve a complete alignment because of the large differences inthe primary structure, eight conserved regions (Fig. 1a–h) aligned.In a multiple alignment of 211 P-type ATPases, the eightconserved regions were found to be conserved throughout thesuperfamily, and were therefore designated to define the core ofthe P-type ATPase superfamily [Axelsen and Palmgren, 1998].When the discussion in this study concerns the amino acidresidues of ATP7A that are part of the present sequence alignment(Fig. 1), it is based on the homology models. When the discussionconcerns an amino acid residue that is not part of the presentsequence alignment, it is based on the work of Axelsen andPalmgren [1998]. Only regions that are conserved betweenATP7A and ATP2A1 are modeled. The residues that are notincluded in the models will in all cases allow for the gaps to befused without geometric strains. We suggest that the essential partsof the models with highly conserved regions (Fig. 1a–c and e–h) inthe A domain and the P domain, including the phosphorylationsite, are fully superimposable on the structures of ATP2A1, whichindicates that the homology models are a good approximation ofthe structural arrangement of ATP7A in these regions. Further-more, despite the relatively low sequence homology in thetransmembrane region (TM) region (Fig. 1d), we suggest thatthe protein is also structurally conserved in this region. This isbased on the assumption that the CPC motif in heavy-metalpumps corresponds to the IPE motif in the calcium pumps, andthat p.P1001 in the CPC motif corresponds to the proline residuethat is invariant among all P-type ATPases. If this assumption iscorrect, the homology models may offer a good approximation forthe structure in the TM region. If the assumption is not correct,the homology models are unlikely to be valid in the TM region.

Immuno£uorescence

Skin fibroblasts obtained from the patients and controls werecultured in a 1:1 mixture of RPMI 1640 (ICN Biomedicals, Inc.;www.mpbio.com) with 20 mM HEPES and a nutrient mixture of

HUMANMUTATION 26(2), 84^93,2005 85

F-10 Ham’s media, supplemented with 7.5% Amnio Max (LieTechnologies) C100 supplement (Life Technologies; www.lifetech.com), 4% fetal calf serum, penicillin, and streptomycin. Forimmunofluorescence, microscopy cells grown on glass coverslipswere incubated in pure F-10 with 50 mM bathocuproine disulfonicacid (BCS; Sigma; www.sigmaaldrich.com) for 2 hr. The cells werefixed in freshly prepared 4% paraformaldehyde in PBS for 20 minat 41C, and washed and permeabilized in 0.2% Triton-X100 inPBS. Nonspecific signals were blocked with 3% BSA in PBS

containing 0.2% Triton-X100 for 30 min, followed by incubationsin the same blocking media containing chicken anti ATP7A(1:1,000; ab13995 from Abcam (www.abcam.com), against theamino acids 1407–1500) + mouse anti GS28 (1:1,000; from BDBioscience; www.bdbiosciences.com) primary antibodies for 2 hr.Alexa 488-conjugated goat anti-chicken (1:2,000) and Alexa 548-conjugated donkey anti-mouse (1:1,000) antibodies (MolecularProbes, Eugene, OR; www.probes.invitrogen.com) were used todetect the primary antibodies (1 hr). Alexa 488-conjugated rabbit

TABLE 1. Comprehensive List of Di¡erent ATP7A/atp7aMissenseMutations Identi¢ed in the Sequence Encoding the Residuesp.V842^p.S1404n

Nucleotide changeaAmino acidchangeb,c Exon

CodonATP2A1b Motif and domaind Region Phenotype

c.2531G4A p.R844H 12 147 RGD, A domain a MODe/classicf

c.2557G4A; c.2557G4C p.G853R 12 156 PGG, A domain a Classicc.2579G4T p.G860V 12 163 A domain a Classicg

p.L873Rh 12 179 LITGEA, A domain b Classicc.2626G4C p.G876R 12 182 TGEA, A domain b Classicc.2627G4A p.G876Ei 13 182 TGEA, A domain b LSc.2771G4A p.Q924R 13 243 Stalk S4 OHSc.2998T4C p.C1000Ri 15 307 CPC,TM6 d LS

p.L1006Pj 15 313 TM6 d LS (treated)c.3020C4T p.A1007V 15 314 TM6 d Mild

p.A1007Tk# 15 313 TM6 atp7aMopew, mildk

c.3044G4A p.G1015D 15 322 Stalk S6 LS/MODp.G1019Dj 15 326 Stalk S6 Classic

c.3131A4G p.D1044G 16 351 DKTG, P domain e Classic (also 5;12translocation)

p.K1045Tl,m# 16 352 DKTG, P domain e atp7aMo vbr, mildl,m

c.3299T4C p.L1100P 17 absent on Figure1,proposedN domain

Classic

p.G1118Dn 17 absent on Figure1,proposedN domain

p.G1255Rn 19 626 TGDN, P domain f Classicc.3844A4G p.K1282Eo 20 684 PSHK, P domain g Classicc.3899G4A p.G1300Ei 20 702 GDGIND, P domain g Classic

p.G1302Ro 20 704 GDGIND, P domain g Classicc.3905G4T p.G1302Vi 20 704 GDGIND, P domain g Classicc.3912T4A p.N1304Ki 20 706 GDGIND, P domain g Classicc.3914A4C p.D1305A 20 707 GDGIND, P domain g Classicc.3943G4A p.G1315R 20 717 ANVG, P domain g Classicc.3974C4T p.A1325V 20 727 GTDVA, P domain h Moo/OHS

p.S1344R; p.I1345Fp 21 746^747 Stalk S7 hp.A1362Dq 21 764 TM7 Mildp.A1362Vr 21 764 TM7 Mild

c.4105G4A p.G1369R 21 771 TM7 Classicp.A1373Dl# 21 774 TM7 atp7aMo11H severe,

die in uterop.S1390Ps# 22 790 TM8 atp7aMoml

c.4190C4T p.S1397F 22 800 TM8 ClassicnFormutations identi¢ed in this study nucleotide change is also shown.aThemutation numbering is based on the cDNA sequence L06133.1; +1corresponds to theA of theATG translation initiation codon.bThe reference sequences used is NP_000043.2 (ATP7A) and P04191 (ATP2A1).The initiationATG codon is codon1.cSubstituted residues inmurine strains aremarkedwith a number sign #.dResidues conserved in P-typeATPases are in bold.eSztriha et al. [1994].The range of clinical spectrum ofMenkes disease is classic4LS4MOD4mild4OHS.fGerdes et al. [1988].gDabadie et al. [1984].hOgawa et al. [1999].iThe amino acid substitution, but not the nucleotide, has previously beenmentioned in a review [Tˇmer et al.,1999].jTˇmer et al. [1997].kLevinson et al. [1997].lCecchi et al. [1997].mReed and Boyd [1997].nHahn et al. [2001].oDas et al. [1994].pGu et al. [2001].qKaler [1998].rAmbrosini andMercer [1999].sMori andNishimura [1997]; Murata et al. [1997];Ohta et al. [1997].MOD, moderate; LS, long survival classic (46 year); OHS, occipital horn syndrome.

86 HUMANMUTATION 26(2), 84^93,2005

anti-goat antibodies (1:2,000; Molecular Probes) were subse-quently used to amplify the signal from the goat anti-chickensecondary antibodies (1 hr). The coverslips were finally mountedin Dapi antifade cytocell. Skin fibroblasts with the ATP7Amutation c.121-?-8333+?del (deletion of exon 3-23) wereincluded as negative control cells, and fibroblasts from a normalperson were used as positive control fibroblasts.

RESULTSIdenti¢cation of Missense Mutations in theATP7AGene

We identified 21 missense mutations, leading to 20 separateamino acid changes (Table 1), in the part of ATP7A that encodesthe amino acid stretch p.V842–p.S1404. Together with ninemutations reported earlier [Das et al., 1994; Tumer et al., 1997;Levinson et al., 1997; Kaler 1998; Ambrosini and Mercer, 1999;Ogawa et al., 1999; Gu et al., 2001; Hahn et al., 2001] and fourmutations in the murine homologue, atp7a [Cecchi et al., 1997;Levinson et al., 1997; Mori and Nishimura, 1997; Murata et al.,1997; Ohta et al., 1997; Reed and Boyd, 1997], a total number of34 missense mutations are known to date in the part of ATP7A/atp7a sequence encoding residues p.V842–p.S1404. The totalnumber of substituted residues in this region is 30 (Table 1). Thefour codons encoding p.G876, p.A1007, p.G1302, and p.A1362have all been affected by two different mutations (Table 1).

Homology Between ATP7A and ATP2A1

To enable a 3D structural alignment of ATP7A to ATP2A1, weperformed a sequence alignment of the two ATPases (seeMaterials and Methods; Fig. 1). In contrast to ATP2A1, the N-terminal part of ATP7A consists of six repetitive cytoplasmiccopper-binding domains (amino acid positions 6-77, 169-240, 275-346, 375-446, 486-557, and 562-633, respectively). The sixcopper-binding domains are followed by two predicted TM helices(amino acid positions 654-675 and 715-734) [Axelsen andPalmgren, 1998]. These helices are conserved in family 1B(including ATP7A), but are not present in family 2B (includingATP2A1) (Fig. 2). The structural homology between ATP7A andATP2A1 is believed to start at TM3 (about codon 740) inATP7A, and at TM1 in ATP2A1 (about codon 48), although thesequence homology between the two sequences is too low to allowfor a sensible sequence alignment in this region. However, thestructural similarities between the two pumps were sufficient toallow alignment of the major part of the sequence from p.V842 top.S1404 (Fig. 1). Of 338 residues that are aligned between ATP7Aand ATP2A1, 98 are identical in the two sequences, giving anoverall sequence identity of 29.0% in the aligned regions. In theconserved regions (Fig. 1a–h), which define the core of the P-typeATPase superfamily (see Materials and Methods) [Axelsen andPalmgren, 1998], the overall sequence identity is 34.9%, rangingfrom 52% in region g (24 identical out of 46 aligned) to 19% (5/26) in region c.

Localization of AminoAcid Substitutions in thePrimary Structure of ATP7A

Interestingly, when we analyzed the positions of the substitutedresidues in the primary ATP7A/atp7a amino acid sequence (Fig. 1,Table 1), we found that 28 of 30 disease-causing mutationsaffected residues in the most homologous regions of ATP7A andATP2A1 (Fig. 1). Besides the 20 substitutions that affectedresidues located in the eight conserved regions (Fig. 1a–h), eight

mutations affected residues located in other homologous regions.Only two substituted residues (p.L1100P and p.G1118D) werelocated in nonhomologous segments, in the proposed N domain(Table 1).

Suggested Homology Model of ATP7A

Our model covers the region with the highest sequencehomology between ATP7A and ATP2A1, starting within the Adomain (p.V842 in ATP7A corresponding to p.I145 in ATP2A1)and ending with TM8 (p.S1404 in ATP7A, p.L807 in ATP2A1)(Fig. 1). The N domain has not been modeled, because of lowsequence homology (the corresponding primary sequence has beenomitted from the sequence alignment, indicated by a vertical linein the figure (Fig. 1)), and the remaining gaps in the model areindicated by a yellow color in the sequence alignment. Allconserved regions (Fig. 1a–h) [Axelsen and Palmgren, 1998] areat least partially covered by the homology model. The conserva-tion of the invariant proline in region d (p.P1001 in the CPCmotif; Fig. 1) suggests that the partly unwound alpha helix foundin ATP2A1, which forms the Ca2+ binding site II, is structurallyconserved in ATP7A. This enables us to align region d. Also, thehighly conserved DKTGT consensus motif in region e, whichcontains the phosphorylated p.D1044, is conserved in bothATPases and is likely to have the same structure.

In ATP2A1, the N-terminal region preceding TM1 contains acalcium-binding EF-hand motif, which forms part of the A domain.The corresponding region between TM2 and TM3 in ATP7A istoo short to form such a motif, but we cannot exclude thepossibility that one of the Cu-binding domains may interact withthe A domain instead (Fig. 2).

Location of Altered Residues in theModel of ATP7A

All substituted amino acid residues are mapped in the modeledpart of ATP7A with or without bound Cu+ (Fig. 3A and B,respectively). All residues are more clustered than expected fromthe primary sequence. The altered residues are located near theactive site in the P domain, in the A domain, along the TMhelices, and in the stalk regions (Figs. 3–5). On the basis of thehomology model, we assume that in the absence of Cu+ the TGEmotif in the A domain approaches the TGDN, PSHK, and theGDGIND motifs in the P domain (Figs. 3 and 5). By thismovement, all of the altered residues become located in the centerof the protein.

The P domain, which harbors the catalytic core, has the highestnumber of substituted residues. In the 3D homology modelthe conserved motifs TGDN, PSHK, and GDGIND are locatedclose to the phosphorylation site DKTGT (Fig. 4), and missensemutations in MD patients affecting all these motifs have beenidentified (Table 1). Although the P domain is made up of twoparts separated by about 178 amino acid residues, the substitutedresidues are clustered together on the cytoplasmic surface in theATP7A model (Figs. 3 and 4).

In ATP2A1 the A domain is almost isolated, connected to theTM region by loops [Toyoshima et al., 2000]. There is noalignment between the N-terminal portions of the A domain inATP7A and ATP2A1, but the region between TM4 and TM5 ofATP7A, corresponding to the residues from about p.V842 top.E921, aligned well with the region between TM2 and TM3 inATP2A1. In this region five residues are substituted as a result ofsix different MD mutations (Fig. 1, Table 1).

HUMANMUTATION 26(2), 84^93,2005 87

Detection and Localization of ATP7A in MenkesFibroblast

Mutations can abrogate function and affect the proteinsynthesis, stability, or localization. In an attempt to distinguishbetween these possibilities, we used immunofluorescence toanalyze fibroblasts obtained from the different patients for thepresence and localization of ATP7A protein. The cells were

treated with a low concentration of BCS for 2 hr, as this leads to aTGN localization of ATP7A in the positive control fibroblasts andmakes the signal more distinct (unpublished results). We analyzedfibroblasts containing the mutations p.R844H, p.G860V, p.G76R,p.Q924R, p.C1000R, p.A1007V, p.G1015D, p.D1044G,p.K1282E, p.G1300E, p.G1302V, p.N1304K, p.D1305A,p.G1315R, p.A1325V, p.G1369R, and p.S1397F. Unfortunately,it was not possible to obtain fibroblasts from patients carrying the

FIGURE 1. Linear alignment of conserved regions of ATP2A1 and ATP7A.The amino acid residues that are conserved in 35 respec-tively 49 out of 49 copperP-typeATPases are shown in lines 4 and 5.The correspondingATP2A1sequence is shown in the sixth line.The coloring refers to the proposed domain structure of ATP7A, based on the results from the crystal structures of the calciumpump[Toyoshima et al., 2000]. Green indicates theA domain, pink the P domain, dark blue the TM, and light blue the stalk regions.Thesequencehighlightedwith yellowcolor in the alignment represents all stretches of the sequence that were notmodeled, including theN domain.The last line of the alignment shows the consensus sequence in 90 of159 P-typeATPases (including all subfamilies).Theconsensus amino acids shown in lowercase letters arepositionswhere the sequences haveoneof two conserved amino acids (I orV, Dor E, and ForY).The numbers above the sequences refer toATP7A, and the numbers below the sequences refer toATP2A1.The ver-tical line after residue1064 inATP7A illustrates omitted residues, all of which are located in the proposedN domain. Missensemuta-tions identi¢ed inATP7A (human) or atp7a (mouse) are indicated in the second and the ¢rst line, respectively, of the alignment. Allconsensus sequences are according toAxelsen and Palmgren [1998].The reference sequences used are NP_000043.2 (ATP7A) andP04191 (ATP2A1).The initiationATG codon is codon1.

88 HUMAN MUTATION 26(2), 84^93,2005

FIGURE 2. Domain organization of ATP7A and ATP2A1. Schematic pictures of the two pumps based on the crystal structure ofATP2A1 [Toyoshimaet al.,2000].Green indicates theAdomain, pink thePdomain, yellow theNdomain, darkblue the TM, and lightblue the stalk regions (S).The six copper-bindingdomains,which areunique for the copperATPases, are in orange.Thepositions andsequencemotifs conserved among theATPases are shown.The coloring corresponds to the coloring of the alignment (Fig.1).

FIGURE 3. Homology models of ATP7A in the absence of Cu+ ions (A) or presence of Cu+ ions (B).The structural locations of theconservedmotifs arehighlighted in red.OneCu+ ionbetweenTM6 andTM8 appears as a red sphere (B).The locations of themutatedresidues are illustrated as spheres. Green indicates theA domain, pink P domain, dark blue the TM, and light blue the stalk regions.The asterisk (*) illustrates the positionof theN domain that is not included in themodeling.Thecoloringcorresponds to the coloringof the alignment (Fig.1).

FIGURE 4. A¡ected residues in thePdomain.The locations of a¡ected residues in thePdomainofATP7A in the absenceofCu+ (A) orpresence of Cu+ (B) are shown.The mutated residues are illustrated in a ball-and-stick representation.The mutated p.N1304 andp.G1315 are omitted from the ¢gure for simplicity.

HUMAN MUTATION 26(2), 84^93,2005 89

mutations p.G853R (c.2557G>A and c.2557G>C), p.G876E, orp.L1100P. Immunofluorescence microscopy revealed that in thepositive control and in 10 of 17 analyzed Menkes fibroblastcultures, the perinuclear localization of ATP7A protein variantswas normal, which suggests that these 10 mutations causeddysfunction at the structural level. These ATP7A protein variantscolocalized with GS28, a 28-kDa SNARE protein located withinthe Golgi compartment (Fig. 6) [Subramaniam et al., 1996].No ATP7A signal was detected in the negative control cells

(Fig. 6d–f). Slightly different patterns were seen in the fibroblastscontaining the p.Q924R and p.G1302V mutations, since thesignals of ATP7A and GS28 did not completely overlap (Fig. 6j,q).In addition to staining in the perinuclear region of the cells, therewas also ATP7A staining extending into cytoplasmic regions. NoATP7A signal was observed in the five cultures containing themutations p.R844H, p.G860V, p.G876R, p.A1325V, andp.G1369R, indicating that the lack of activity reflected either afailure to synthesize the protein or very rapid degradation.

FIGURE 5. Inter domain conformation of the A and P domains. A fraction of the P domain is shown in red, and a fraction of the Adomain is shown in green. Intermolecular localization of the two domains is shown in the Cu+-free (A) vs. the Cu+-bound form (B).A¡ected residues are shown in a ball-and- stick representation. Invariant motifs are indicated.

FIGURE 6. Detection ofATP7A in patient ¢broblasts. Immuno£uorescencemicroscopywasused to analyze patients’cells for the pre-sence of ATP7A protein.The cells were cultured for 2 hr in media containing BCS before they were processed for double-labeledimmuno£uorescence using ATP7A and GS28 antibodies. The ATP7A signal alone in the (a) positive and (d) negative controlcells is shown.TheGS28 signal alone in the (b) positive and (e) negative control cells is shown. c, f, and g^x:Combined image fromdouble-labeling of the control cells and the di¡erent patients’ ¢broblasts.

90 HUMAN MUTATION 26(2), 84^93,2005

In fibroblasts from most of the patients with mutations in the Pdomain (p.D1044G, p.K1282E, p.G1300E, p.N1304K, p.D1305A,and p.G1315R), the ATP7A protein variants were localized in thesame perinuclear region (consistent with the TGN) as the wildtype, while no ATP7A protein was detected in the fibroblastscontaining the p.A1325V mutation. In fibroblasts containing thep.G1302V mutation, only a fraction of the variant proteins werecorrectly localized in the TGN. Thus, missense mutations in theP domain can inactivate the protein, effect the synthesis or thestability of the protein variant, or mislocalize it, depending on thesite of the amino acid substitution.

In the analyzed fibroblast cultures obtained from three(p.R844H, p.G860V, and p.G876R) of the six patients with amutation in the A domain, no ATP7A signal could be identified(Fig. 6). These disease-causing mutations appear to affect thesynthesis of the protein or destabilize it. How the other mutationsidentified in the A domain affect the level or localization of theATP7A protein we do not know.

The ATP7A variants with the TM mutations p.C1000A,p.L1006P, p.A1007V, or p.S1397F are correctly localized to theperinuclear region but are nonfunctional. In contrast, no ATP7Asignal was observed in the p.G1369R cell line.

DISCUSSION

Twenty-eight of 30 disease-causing mutations affect residues inthe most conserved regions of ATP7A. All residues are moreclustered in the 3D model of the protein than was expected fromthe primary sequence.

Most of the analyzed mutations (10 of 17) do not affect thesynthesis, stability, or localization of the ATP7A protein.Presumably, these mutations are ‘‘true’’ functional mutations.Two mutations lead to a partially correct localization. Although afraction of the protein variants appears to be correctly localized inthe TGN, there was also staining into cytoplasmic regions. For fivemutations that do not lead to a visible protein product, it isunknown whether the effect is due to an inhibition at thetranscription or translation level, or to degradation of the protein.

Mutations in the PDomain

The P domain has sequence homology with the catalytic coredomain of L-2-haloacid dehydrogenase (HAD), with which itshares all of the above-mentioned conserved motifs. Based on thishomology, a shared catalytic mechanism was proposed for P-typeATPases and HAD [Aravind et al., 1998; Li et al., 1998;Toyoshima et al., 2000]. The aspartate (D) and lysine (K) in theDKTG motif are necessary for the Ca2+-dependent phosphoryla-tion of the aspartate [Maruyama and MacLennan, 1988], and it isassumed that phosphorylation is followed by esterification[Aravind et al., 1998]. In mice, substitution of the conservedlysine (K1045) leads to the mouse mutant atp7amo vbr, whichexhibits a Menkes-like disease, although in a milder form [Cecchiet al., 1997; Reed and Boyd, 1997]. The p.D1044G mutation wasidentified in a patient suffering from classic MD (Table 1).

The TGDN motif has been shown to be critical for ATP bindingand catalysis. In the presence of Mg2+ and Ca2+, the ATP bindingis almost completely blocked in the ATP2A1 p.G626A mutant[McIntosh et al., 2004]. The p.D627E mutant is able to bind ATP,but the dephosphorylation rate is reduced relative to the wild type[Clarke et al., 1990; McIntosh et al., 2004]. The correspondingsubstitution p.G1255R has been identified in a classic MD patient[Hahn et al., 2001].

The positive charge of the lysine (K) in PSHK is essential toform a phosphorylated intermediate, and substitutions of thecorresponding lysine in ATP2A1, K684A, K684H, and K684Qlead to an ATPase that is unable to form a phosphorylatedintermediate, while the K684R substitution leads to an ATPasethat does form a phosphorylated intermediate [Vilsen et al., 1992].The positive charge of the lysine (K) is assumed to be importantfor the stabilization of the negative charge of the reactionintermediates during the phosphorylation of D1044. We haveidentified a substitution of the lysine (p.K1282E; Table 1) in apatient with classic MD, which is consistent with the importanceof this residue.

The conserved GDGIND is important for Mg2+ binding,phosphorylation, and phosphoenzyme hydrolysis [Vilsen et al.,1991; Pedersen et al., 2000; Voskoboinik et al., 2001]. Theconserved aspartates (D) in GDGIND are assumed to be involvedin the delivery of water for acyl-phosphate intermediate hydrolysis.Four missense mutations affecting four of the five conservedresidues in the GDGIND motif were identified in classic MDpatients, which stress the importance of this motif (Table 1).

Mutations in theTMSegments

The copper-binding domains are essential for the copper-stimulated trafficking of ATP7A from TGN to the plasmamembrane [Strausak et al., 1999], and the engineered mutationof all copper-binding sites results in a copper-accumulatingphenotype [Voskoboinik et al., 1999]. Nevertheless, it has beenproposed that the N-terminal domain is not essential for the ATP-dependent copper translocation activity. Using an in vitro vesicleassay, Voskoboinik et al. [1999, 2001] showed that mutation of allsix copper-binding sites still leaves 55% of the wild-typetranslocation activity. In line with this, a functional analysis ofthe chimeric proteins of Wilson ATP7B and ZntA, a Pb/Zn/CdATPase from E. coli, demonstrated that the TM segmentsdetermine the metal specificity [Hou et al., 2001].

Assuming that the CPC motif in heavy-metal pumps corre-sponds to the IPE motif in the ATP2A1, we suggest that thebackbone carbonyls of p.C997, p.I998, and p.C1000, and the sitechains of p.C1002, p.M1393, and p.S1397 may be important forbinding and transport of Cu+. These residues correspond top.V304, p.A305, p.I307, p.E309, p.N796, and p.D800 in ATP2A1,which comprise the Ca2+ binding-site II in ATP2A1. In amutational study of Ccc2p, the yeast Menkes/Wilson homologue,it was suggested that the cysteine corresponding to p.C1000 isnecessary for copper dissociation, whereas p.C1002 has a role incopper binding [Lowe et al., 2004]. Both p.M1393 and p.S1397are specific to the subfamily 1B-1 of copper ATPases [Arguello2003], which suggests that they are important for metal specificity.Also, p.L1006 and p.A1007 are relatively conserved among thesubgroup of copper ATPases [Arguello, 2003].

CONCLUSIONS

A large number of mutagenesis studies have been performed toclarify the function of selected amino acid residues in the ATPases.The resulting effect is usually measured in terms of the effect oncation translocation and ATPase or phosphatase activity. However,since binding of Cu+ is required to activate phosphoryl transferfrom ATP to the phosphorylation site, it is often difficult todistinguish whether a specific mutation affects the ATP hydrolysesdirectly or indirectly by affecting the pathway or binding sites ofthe cations. Furthermore, since most studies are performed in vitroor on nonhuman organisms, the results may not always reflect the

HUMANMUTATION 26(2), 84^93,2005 91

effect in humans. Analysis of the structural location of disease-causing mutations, as enabled by the current model, is a usefulalternative. However, this model is only the first approximation ofpossible structural arrangements in ATP7A, and future experi-ments will be necessary to determine whether all of the presentfindings hold true.

A relatively mild Menkes phenotype is observed mainly whenmutations affect residues in the stalk regions or TM domains. Incontrast, all of the human mutations (except one) that affectresidues located in the P domain were identified in patientssuffering from classic MD. The only exception was the p.A1325Vmutation, which has been identified in a family with a varyingphenotype [Borm et al., 2004]. In contrast, there does not appearto be any clear correlation between the localization of the ATP7Aprotein and the phenotype. The patients with no visible amount ofATP7A represent classic or modified MD, and the patients withcorrectly localized ATP7A represent all types, from mild OHS tosevere classic MD.

ACKNOWLEDGMENTS

We thank all of the patients and their families who participatedin this study, and the clinicians who referred their patients to theJohn F. Kennedy Institute.

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