Structure of the Alzheimer's Disease Amyloid Precursor Protein Copper Binding Domain
2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês
10.1074/jbc.m300629200
ISSN1083-351X
AutoresKevin J. Barnham, William J. McKinstry, Gerd Multhaup, Denise Galatis, Craig J. Morton, Cyril C. Curtain, Nicholas A. Williamson, Anthony R. White, Mark G. Hinds, Raymond S. Norton, Konrad Beyreuther, Colin L. Masters, Michael W. Parker, Roberto Cappai,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoA major source of free radical production in the brain derives from copper. To prevent metal-mediated oxidative stress, cells have evolved complex metal transport systems. The Alzheimer's disease amyloid precursor protein (APP) is a major regulator of neuronal copper homeostasis. APP knockout mice have elevated copper levels in the cerebral cortex, whereas APP-overexpressing transgenic mice have reduced brain copper levels. Importantly, copper binding to APP can greatly reduce amyloid औ production in vitro. To understand this interaction at the molecular level we solved the structure of the APP copper binding domain (CuBD) and found that it contains a novel copper binding site that favors Cu(I) coordination. The surface location of this site, structural homology of CuBD to copper chaperones, and the role of APP in neuronal copper homeostasis are consistent with the CuBD acting as a neuronal metallotransporter. A major source of free radical production in the brain derives from copper. To prevent metal-mediated oxidative stress, cells have evolved complex metal transport systems. The Alzheimer's disease amyloid precursor protein (APP) is a major regulator of neuronal copper homeostasis. APP knockout mice have elevated copper levels in the cerebral cortex, whereas APP-overexpressing transgenic mice have reduced brain copper levels. Importantly, copper binding to APP can greatly reduce amyloid औ production in vitro. To understand this interaction at the molecular level we solved the structure of the APP copper binding domain (CuBD) and found that it contains a novel copper binding site that favors Cu(I) coordination. The surface location of this site, structural homology of CuBD to copper chaperones, and the role of APP in neuronal copper homeostasis are consistent with the CuBD acting as a neuronal metallotransporter. Alzheimer's disease amyloid precursor protein copper binding domain electron paramagnetic resonance Protein Data Bank heteronuclear single quantum correlation Alzheimer's disease (AD)1 is characterized by progressive neuronal dysfunction, reactive gliosis, and the formation of amyloid plaques in the brain. The cause of the neuronal cell loss in AD is unclear but may be related to increased oxidative stress from excessive free radical generation (1Martins R.N. Harper C.G. Stokes G.B. Masters C.L. J. Neurochem. 1986; 46: 1042-1045Crossref PubMed Scopus (204) Google Scholar, 2Smith M.A. Richey Harris P.L. Sayre L.M. Beckman J.S. Perry G. J. Neurosci. 1997; 17: 2653-2657Crossref PubMed Google Scholar, 3Bush A.I. Curr. Opin. Chem. Biol. 2000; 4: 184-191Crossref PubMed Scopus (693) Google Scholar, 4Sayre L.M. Perry G. Harris P.L. Liu Y. Schubert K.A. Smith M.A. J. Neurochem. 2000; 74: 270-279Crossref PubMed Scopus (461) Google Scholar). A major source of free radical production in the brain is from the transition metals copper and iron (3Bush A.I. Curr. Opin. Chem. Biol. 2000; 4: 184-191Crossref PubMed Scopus (693) Google Scholar, 5Sayre L.M. Perry G. Smith M.A. Curr. Opin. Chem. Biol. 1999; 3: 220-225Crossref PubMed Scopus (206) Google Scholar). These metals are vital for life because of their high redox activity and have been utilized in a number of enzymatic pathways, including cellular respiration. However, if the redox reactivity of copper and iron is not strictly regulated, this can result in the generation of toxic reactive oxygen intermediates (2Smith M.A. Richey Harris P.L. Sayre L.M. Beckman J.S. Perry G. J. Neurosci. 1997; 17: 2653-2657Crossref PubMed Google Scholar). The potential for oxidative damage from reactive oxygen intermediates in the aging brain is further enhanced by the high oxygen consumption and relatively low antioxidant levels in brain tissue. To prevent transition metal-mediated oxidative stress, cells have evolved elaborate systems for copper storage and transport that deliver copper and iron to metalloenzymes and proteins. A number of studies have implicated cell surface metalloreductases in the reduction of Cu(II) to Cu(I), which is the form of the metal ion that is delivered to the cytoplasm of eukaryotic cells via copper transporters (6Puig S. Thiele D.J. Curr. Opin. Chem. Biol. 2002; 6: 171-180Crossref PubMed Scopus (570) Google Scholar). To avoid Cu(I) redox chemistry inside the cell, Cu(I) ions are escorted by specific cytosolic metalloproteins such as the copper chaperones that are involved in intracellular copper trafficking to Wilson's disease copper ATPase and the copper/zinc superoxide dismutase (7Waggoner D.J. Bartnikas T.B. Gitlin J.D. Neurobiol. Dis. 1999; 6: 221-230Crossref PubMed Scopus (754) Google Scholar). This results in unbound copper being essentially absent in the intracellular environment (8Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1346) Google Scholar). Therefore, cupro-proteins play an important role in maintaining cellular copper metabolism (9Andrews N.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6543-6545Crossref PubMed Scopus (27) Google Scholar).Both APP and Aऔ, a proteolytic breakdown product of APP, play a central role in Alzheimer's disease and can strongly bind Cu(II) (Kd APP ≈ 10 nm) and reduce it to Cu(I) in vitro (10Hesse L. Beher D. Masters C.L. Multhaup G. FEBS Lett. 1994; 349: 109-116Crossref PubMed Scopus (221) Google Scholar, 11Multhaup G. Schlicksupp A. Hesse L. Beher D. Ruppert T. Masters C.L. Beyreuther K. Science. 1996; 271: 1406-1409Crossref PubMed Scopus (580) Google Scholar, 12Atwood C.S. Moir R.D. Huang X. Scarpa R.C. Bacarra N.M. Romano D.M. Hartshorn M.A. Tanzi R.E. Bush A.I. J. Biol. Chem. 1998; 273: 12817-12826Abstract Full Text Full Text PDF PubMed Scopus (934) Google Scholar, 13Cherny R.A. Legg J.T. McLean C.A. Fairlie D.P. Huang X. Atwood C.S. Beyreuther K. Tanzi R.E. Masters C.L. Bush A.I. J. Biol. Chem. 1999; 274: 23223-23228Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 14Huang X. Atwood C.S. Hartshorn M.A. Multhaup G. Goldstein L.E. Scarpa R.C. Cuajungco M.P. Gray D.N. Lim J. Moir R.D. Tanzi R.E. Bush A.I. Biochemistry. 1999; 38: 7609-7616Crossref PubMed Scopus (1020) Google Scholar, 15Huang X. Cuajungco M.P. Atwood C.S. Hartshorn M.A. Tyndall J.D. Hanson G.R. Stokes K.C. Leopold M. Multhaup G. Goldstein L.E. Scarpa R.C. Saunders A.J. Lim J. Moir R.D. Glabe C. Bowden E.F. Masters C.L. Fairlie D.P. Tanzi R.E. Bush A.I. J. Biol. Chem. 1999; 274: 37111-37116Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar). The APP copper binding domain (CuBD) is located in the N-terminal cysteine-rich region next to the growth factor-like domain (10Hesse L. Beher D. Masters C.L. Multhaup G. FEBS Lett. 1994; 349: 109-116Crossref PubMed Scopus (221) Google Scholar, 16Rossjohn J. Cappai R. Feil S.C. Henry A. McKinstry W.J. Galatis D. Hesse L. Multhaup G. Beyreuther K. Masters C.L. Parker M.W. Nat. Struct. Biol. 1999; 6: 327-331Crossref PubMed Scopus (206) Google Scholar) (Fig. 1). APP is a member of a multigene family, and the CuBD sequence is similar among the different APP family paralogs and orthologs, suggesting an overall conservation in its function or activity. In vivo studies show that APP expression is a key modulator of neuronal copper homeostasis since APP knockout mice have increased copper levels in the brain (17White A.R. Reyes R. Mercer J.F.B. Camakaris J. Zheng H. Bush A.I. Multhaup G. Beyreuther K. Masters C.L. Cappai R. Brain Res. 1999; 842: 439-444Crossref PubMed Scopus (255) Google Scholar). Conversely, APP overexpressing transgenic mice have significantly reduced copper levels in transgenic mouse brain (18Maynard C.J. Cappai R. Volitakis I. Cherny R.A. White A.R. Beyreuther K. Masters C.L. Bush A.I. Li Q.X. J. Biol. Chem. 2002; 277: 44670-44676Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). The APP CuBD can also modulate Cu(I)-mediated neurotoxicity (19White A.R. Multhaup G. Maher F. Bellingham S. Camakaris J. Zheng H. Bush A.I. Beyreuther K. Masters C.L. Cappai R. J. Neurosci. 1999; 19: 9170-9179Crossref PubMed Google Scholar) and, depending on the ortholog, can either promote or inhibit copper neurotoxicity (20White A.R. Multhaup G. Galatis D. McKinstry W.J. Parker M.W. Pipkorn R. Beyreuther K. Masters C.L. Cappai R. J. Neurosci. 2002; 22: 365-376Crossref PubMed Google Scholar). The interaction between the APP-Cu(I) species with hydrogen peroxide can result in Cu(I) oxidation to Cu(II) and APP fragmentation (21Multhaup G. Ruppert T. Schlicksupp A. Hesse L. Eckhard E. Pipkorn R. Masters C.L. Beyreuther K. Biochemistry. 1998; 37: 7224-7230Crossref PubMed Scopus (135) Google Scholar). Of importance to Alzheimer disease pathology is the finding that increasing the copper concentration modulates APP processing, resulting in greatly reduced Aऔ production and increased levels of the cell-bound and secreted forms of APP (22Borchardt T. Camakaris J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Biochem. J. 1999; 344: 461-467Crossref PubMed Scopus (158) Google Scholar, 23Borchardt T. Schmidt C. Camarkis J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Cell. Mol. Biol. (Noisy-le-Grand). 2000; 46: 785-795PubMed Google Scholar). Mutagenesis of histidine residues within CuBD inhibits the effects of copper on APP expression and proteolysis (23Borchardt T. Schmidt C. Camarkis J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Cell. Mol. Biol. (Noisy-le-Grand). 2000; 46: 785-795PubMed Google Scholar).The importance of copper to Alzheimer's disease is emphasized by the neurotoxic interaction between the Aऔ peptide and copper. The Aऔ peptide binds copper with a high affinity and reduces Cu(II) to Cu(I), resulting in the catalytic generation of hydrogen peroxide (H2O2) and Aऔ aggregation (24Opazo C. Huang X. Cherny R.A. Moir R.D. Roher A.E. White A.R. Cappai R. Masters C.L. Tanzi R.E. Inestrosa N.C. Bush A.I. J. Biol. Chem. 2002; 277: 40302-40308Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). Aऔ and copper can interact to form an oligomeric complex that binds copper at a copper/zinc superoxide dismutase-like binding site (25Curtain C.C. Ali F. Volitakis I. Cherny R.A. Norton R.S. Beyreuther K. Barrow C.J. Masters C.L. Bush A.I. Barnham K.J. J. Biol. Chem. 2001; 276: 20466-20473Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). The importance of copper in AD pathology has been demonstrated by the ability of a chelator (clioquinol) to disaggregate amyloid bothin vitro and in a transgenic mouse model in vivo(26Cherny R.A. Atwood C.S. Xilinas M.E. Gray D.N. Jones W.D. McLean C.A. Barnham K.J. Volitakis I. Fraser F.W. Kim Y. Huang X. Goldstein L.E. Moir R.D. Lim J.T. Beyreuther K. Zheng H. Tanzi R.E. Masters C.L. Bush A.I. Neuron. 2001; 30: 665-676Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar). It is plausible that copper binding to CuBD and Aऔ are linked phenomenon. The modulation of copper levels by the APP CuBD would influence Aऔ-Cu interactions, resulting in increased H2O2 from Aऔ-Cu or an overall increase in neuronal reactive oxygen intermediate production. Changes in copper levels will also affect APP processing into Aऔ, thus controlling the production of neurotoxic Aऔ. Therefore, defining the interaction of copper with APP has important consequences for Aऔ production and AD pathogenesis and subsequent therapeutic intervention. To understand the interaction of copper with the APP CuBD at the molecular level, we have determined the three-dimensional structure of the CuBD (APP residues 124–189) by NMR spectroscopy. The structure has led to the identification of a novel copper binding site. The CuBD has structural homology to copper chaperones, thus suggesting the APP CuBD functions as a neuronal metallotransporter and/or metallochaperone.DISCUSSIONAPP has a copper binding domain located in the N-terminal cysteine-rich region that can strongly coordinate Cu(II) and reduce it to Cu(I) (Fig. 1). It has been demonstrated that this domain can modulate copper homeostasis and production of Aऔ, a peptide that plays a central role in the progression of Alzheimer's disease. Here we report the structure of this domain and identify the residues (His-147, His-151, Tyr-168, Met-170) involved in coordinating copper and the possible mechanism for copper reduction. The nature and orientation of these residues constitute a novel copper binding site.His-147 and His-151 were shown previously to be necessary for copper binding (11Multhaup G. Schlicksupp A. Hesse L. Beher D. Ruppert T. Masters C.L. Beyreuther K. Science. 1996; 271: 1406-1409Crossref PubMed Scopus (580) Google Scholar). The orientation of these residues in the three-dimensional structure indicates that, with very small side-chain movements, a tetrahedral metal binding site suitable for coordinating Cu(I) is formed (Fig. 4). Such a site is reminiscent of the blue copper proteins that bind copper with a tetrahedral arrangement of ligands consisting of two histidines, a methionine, and a cysteine residue (36Adman E.T. Anfinsen C.B. Edsall J.T. Richards F.M. Eisenberg D.S. Adv. Protein Chem. 42. Academic Press Inc., San Diego, CA1991: 145-197Google Scholar). The binding site in CuBD appears novel; a search of the PDB failed to identify a copper site with the same ligands. The closest example was peptidylglycine monoxygenase (PDB code 1PHM), which contains a redox active Cu(II) binding site consisting of two histidine residues, a methionine residue, and a water molecule in a tetrahedral coordination about the metal (37Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Science. 1997; 278: 1300-1305Crossref PubMed Scopus (303) Google Scholar). Beyond this, there was no sequence or structural similarities between the two proteins.The coordination of Cu(II) to the tetrahedrally arranged His-147, His-151, Tyr-168, and Met-170 (Fig. 4) can explain the redox chemistry associated with Cu binding to APP. In general, four coordinate Cu(II) ions favor a square planar coordination sphere about the metal, whereas Cu(I) generally prefers a tetrahedral arrangement (38Casella L. Gullotti M. Karlin K.D. Tyeklar Z. Bioinorganic Chemistry of Copper. Chapman ' Hall, New York1993: 292-305Crossref Google Scholar). The EPR data (Fig. 5) suggest that Cu(II) bound to APP CuBD is distorted away from the square plane toward a tetrahedral structure. Histidine residues are common ligands for Cu(I) sites, and thioether ligands are known to stabilize Cu(I) in model compounds (38Casella L. Gullotti M. Karlin K.D. Tyeklar Z. Bioinorganic Chemistry of Copper. Chapman ' Hall, New York1993: 292-305Crossref Google Scholar). Oxygen ligands are more common in Cu(II) complexes, and an oxygen ligand in stellacyanin is thought to be a major factor in this protein having the lowest reduction potential of all blue proteins (39Hart P.J. Nersissian A.M. Herrmann R.G. Nalbandyan R.M. Valentine J.S. Eisenberg D. Protein Sci. 1996; 5: 2175-2183Crossref PubMed Scopus (169) Google Scholar). Hence, the tyrosine ligand in APP may facilitate binding of Cu(II), and this is subsequently followed by redox reactions. Because the copper binding site of CuBD appears to be a relatively rigid tetrahedral site, Cu(I) binding would be preferred, and the geometry would facilitate the reduction of Cu(II), which in the absence of any exogenous reductants, results in Met-170 oxidization (Fig. 3c). The oxidation of Met-170 in vivo is unlikely because this would alter the characteristics of the binding site, making it less likely to stably bind Cu(I); the presence of exogenous reductants such as ascorbate and thiols would also render metal reduction via Met-170 redundant.Cu(I) sites are normally sequestered inside proteins because exposure could lead to the generation of reactive oxygen species via Fenton chemistry. Indeed such chemistry is observed when copper binds to this domain (21Multhaup G. Ruppert T. Schlicksupp A. Hesse L. Eckhard E. Pipkorn R. Masters C.L. Beyreuther K. Biochemistry. 1998; 37: 7224-7230Crossref PubMed Scopus (135) Google Scholar). The APP copper binding site described here is unusual in that it is surface-exposed but similar to copper chaperone proteins that also possess surface Cu(I) sites (40Poulis T.L. Nat. Struct. Biol. 1999; 6: 709-711Crossref PubMed Scopus (20) Google Scholar). It is thought that the surface location ensures that the metal can be sequestered on binding of the chaperone to its target. Because exposed Cu(I) sites are prone to Fenton chemistry it would seem imperative that copper binding to APP would result in a rapid response. One possible scenario is as follows (Fig. 6). 1) Membrane-bound APP acts as a copper sensor/scavenger (17White A.R. Reyes R. Mercer J.F.B. Camakaris J. Zheng H. Bush A.I. Multhaup G. Beyreuther K. Masters C.L. Cappai R. Brain Res. 1999; 842: 439-444Crossref PubMed Scopus (255) Google Scholar, 18Maynard C.J. Cappai R. Volitakis I. Cherny R.A. White A.R. Beyreuther K. Masters C.L. Bush A.I. Li Q.X. J. Biol. Chem. 2002; 277: 44670-44676Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Cu(II) binding to APP leads to Cu(II) reduction since the CuBD binding site is optimized for Cu(I) binding. 2) The Cu(I) binding signals APP processing or proteolytic breakdown via the non-amyloidogenic route (21Multhaup G. Ruppert T. Schlicksupp A. Hesse L. Eckhard E. Pipkorn R. Masters C.L. Beyreuther K. Biochemistry. 1998; 37: 7224-7230Crossref PubMed Scopus (135) Google Scholar, 22Borchardt T. Camakaris J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Biochem. J. 1999; 344: 461-467Crossref PubMed Scopus (158) Google Scholar). The signal transduction pathway could be triggered by conformational changes or oligomerization caused by the reduction. This is supported by experimental evidence showing that copper binding causes such changes (10Hesse L. Beher D. Masters C.L. Multhaup G. FEBS Lett. 1994; 349: 109-116Crossref PubMed Scopus (221) Google Scholar); our metal binding experiments were accompanied by varying degrees of protein aggregation, and APP oligomerization plays a major role in APP processing (41Scheuermann S. Hambsch B. Hesse L. Stumm J. Schmidt C. Beher D. Bayer T.A. Beyreuther K. Multhaup G. J. Biol. Chem. 2001; 276: 33923-33929Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). 3) The release of the APP ectodomain from the membrane (21Multhaup G. Ruppert T. Schlicksupp A. Hesse L. Eckhard E. Pipkorn R. Masters C.L. Beyreuther K. Biochemistry. 1998; 37: 7224-7230Crossref PubMed Scopus (135) Google Scholar, 22Borchardt T. Camakaris J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Biochem. J. 1999; 344: 461-467Crossref PubMed Scopus (158) Google Scholar) would allow this secreted form to transport the metal to a nearby copper transporter/receptor or for excretion from the body via the liver. This hypothesis would explain the need for a surface location of the Cu(I) ion and provide a molecular basis for the observed role of APP in copper homeostasis (17White A.R. Reyes R. Mercer J.F.B. Camakaris J. Zheng H. Bush A.I. Multhaup G. Beyreuther K. Masters C.L. Cappai R. Brain Res. 1999; 842: 439-444Crossref PubMed Scopus (255) Google Scholar, 18Maynard C.J. Cappai R. Volitakis I. Cherny R.A. White A.R. Beyreuther K. Masters C.L. Bush A.I. Li Q.X. J. Biol. Chem. 2002; 277: 44670-44676Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar) and copper modulation of APP processing (22Borchardt T. Camakaris J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Biochem. J. 1999; 344: 461-467Crossref PubMed Scopus (158) Google Scholar, 23Borchardt T. Schmidt C. Camarkis J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Cell. Mol. Biol. (Noisy-le-Grand). 2000; 46: 785-795PubMed Google Scholar).Figure 6A model outlining the mechanism and biological consequences of the CuBD-copper interaction. APP is a transmembrane molecule that can be cleaved via the amyloidogenic pathway by औ- and γ-secretase cleavage to release Aऔ. Alternatively, cleavage can occur via the non-amyloidogenic pathway by α- and γ-secretase cleavage to release P3 (truncated Aऔ) (49Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (10796) Google Scholar). In the depicted model the key steps are as follows. 1) APP will bind copper via the CuBD (hatched box) in response to copper levels in the extracellular milieu and/or inside the cell. 2) The CuBD, as shown by its structure, will favor the reduction the reduction of Cu(II) to Cu(I) (11Multhaup G. Schlicksupp A. Hesse L. Beher D. Ruppert T. Masters C.L. Beyreuther K. Science. 1996; 271: 1406-1409Crossref PubMed Scopus (580) Google Scholar) and subsequent APP dimerization (41Scheuermann S. Hambsch B. Hesse L. Stumm J. Schmidt C. Beher D. Bayer T.A. Beyreuther K. Multhaup G. J. Biol. Chem. 2001; 276: 33923-33929Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Whether copper participates directly in the dimerization is not known. We would predict other proteins involved in APP processing (secretases or co-factors) could also bind to the APP·Cu(I) complex. 3) The APP·Cu(I) complex promotes the processing of APP via the non-amyloidogenic pathway (22Borchardt T. Camakaris J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Biochem. J. 1999; 344: 461-467Crossref PubMed Scopus (158) Google Scholar), resulting in the secretion of α-secretase-cleaved APP·Cu(I) and the P3 peptide. This results in a decrease in Aऔ levels and an increase in α-secretase-cleaved APP (22Borchardt T. Camakaris J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Biochem. J. 1999; 344: 461-467Crossref PubMed Scopus (158) Google Scholar). The secreted APP·Cu could also be complexed with other molecules/co-factors (4Sayre L.M. Perry G. Harris P.L. Liu Y. Schubert K.A. Smith M.A. J. Neurochem. 2000; 74: 270-279Crossref PubMed Scopus (461) Google Scholar). The secreted APP·Cu(I) can then act as a copper transporter to transport the Cu(I) away from the tissue for excretion via the liver. This is consistent with the in vivoAPP knockout mouse data showing increased copper levels in APP knockout liver and brain (17White A.R. Reyes R. Mercer J.F.B. Camakaris J. Zheng H. Bush A.I. Multhaup G. Beyreuther K. Masters C.L. Cappai R. Brain Res. 1999; 842: 439-444Crossref PubMed Scopus (255) Google Scholar). Alternatively, the APP could act as a copper chaperone and transfer the copper to an as yet unidentified cupro-protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interestingly a search of the Protein Data Bank for similar folds (42Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3552) Google Scholar) yielded 51 structures with the same α-helix packed over a triple strand औ-sheet topology. Three of these proteins are involved in copper chaperone activity including the Menkes copper-transporting ATPase fragment (PDB code 1AW0), metallochaperone Atx1 (PDB code 2U2F), and SOD1 copper chaperone (PDB code 1QUP). These all have a different metal coordination sphere compared with CuBD using two thiol residues in a CXXC motif to bind Cu(I). However, these proteins are intracellular, whereas APP is an extracellular protein with the cysteine residues involved in disulfide bonds and, therefore, are available for metal coordination. Although most copper chaperones identified to date have been shown to utilize the high affinity of the sulfhydryl group of cysteine residues to coordinate Cu(I), it has been reported that CopB copper ATPase, a transmembrane protein fromEnterococcus hirae that is responsible for exporting excess copper, has histidine-rich metal binding motifs (43Cobine P.A. George G.N. Jones C.E. Wickramasinghe W.A. Solioz M. Dameron C.T. Biochemistry. 2002; 41: 5822-5829Crossref PubMed Scopus (112) Google Scholar). The metallochaperone Atx1 displays a number of Lys residues on its surface, and it is thought that these residues play a critical in Atx1 recognizing its partner (Ccc2, a copper transporting P-type ATPase) (44Portnoy M.E. Rosenzweig A.C. Rae T. Huffman D.L. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 15041-15045Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Intriguingly, CuBD also has conserved Lys residues at 155 and 158 that lie in a similar location on the structure as does Lys-24 and -28 for Atx1.The observations that APP knockout mice show specific elevations in brain and liver copper levels (17White A.R. Reyes R. Mercer J.F.B. Camakaris J. Zheng H. Bush A.I. Multhaup G. Beyreuther K. Masters C.L. Cappai R. Brain Res. 1999; 842: 439-444Crossref PubMed Scopus (255) Google Scholar), whereas APP overexpression in mice results in significantly reduced copper levels (18Maynard C.J. Cappai R. Volitakis I. Cherny R.A. White A.R. Beyreuther K. Masters C.L. Bush A.I. Li Q.X. J. Biol. Chem. 2002; 277: 44670-44676Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar) highlights the important role that APP plays in modulating neuronal copper levels. The structure presented here defines how copper interacts with the extracellular region of APP at the atomic level. Modulation of neuronal copper is important because a large body of work has emerged that suggests copper has a significant role to play in a range of neurodegenerative disease (3Bush A.I. Curr. Opin. Chem. Biol. 2000; 4: 184-191Crossref PubMed Scopus (693) Google Scholar) including AD, Creutzfeldt-Jakob disease (45Brown D.R. Sassoon J. Mol. Biotechnol. 2002; 22: 165-178Crossref PubMed Scopus (45) Google Scholar), Parkinson's disease (46Uversky V.N. Li J. Fink A.L. J. Biol. Chem. 2001; 276: 44284-44296Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar), and amyotrophic lateral sclerosis (47Carri M.T. Ferri A. Casciati A. Celsi F. Ciriolo M.R. Rotilio G. Funct. Neurol. 2001; 16: 181-188PubMed Google Scholar).As a possible treatment for Alzheimer's disease it would be highly desirable to develop a drug with specific high affinity binding to APP that would interfere with amyloidogenic APP processingin vivo. The interaction of copper with the CuBD effects APP processing such that Aऔ production is significantly reduced (22Borchardt T. Camakaris J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Biochem. J. 1999; 344: 461-467Crossref PubMed Scopus (158) Google Scholar). This suggests that agonists of copper interaction with APP would have therapeutic potential. The design of such agonists is greatly assisted by the structural information presented here. In addition, the recently reported success in a small-scale phase II clinical trial of the metal chelator clioquinol in reducing Aऔ levels of treated patients illustrates the potential benefits of targeting copper interactions with APP/Aऔ (48Masters, C. L. (2002) Seventh International Geneva/Springfield Alzheimer's Symposium, April 3–6,2000 Geneva, Switzerland.Google Scholar). Alzheimer's disease (AD)1 is characterized by progressive neuronal dysfunction, reactive gliosis, and the formation of amyloid plaques in the brain. The cause of the neuronal cell loss in AD is unclear but may be related to increased oxidative stress from excessive free radical generation (1Martins R.N. Harper C.G. Stokes G.B. Masters C.L. J. Neurochem. 1986; 46: 1042-1045Crossref PubMed Scopus (204) Google Scholar, 2Smith M.A. Richey Harris P.L. Sayre L.M. Beckman J.S. Perry G. J. Neurosci. 1997; 17: 2653-2657Crossref PubMed Google Scholar, 3Bush A.I. Curr. Opin. Chem. Biol. 2000; 4: 184-191Crossref PubMed Scopus (693) Google Scholar, 4Sayre L.M. Perry G. Harris P.L. Liu Y. Schubert K.A. Smith M.A. J. Neurochem. 2000; 74: 270-279Crossref PubMed Scopus (461) Google Scholar). A major source of free radical production in the brain is from the transition metals copper and iron (3Bush A.I. Curr. Opin. Chem. Biol. 2000; 4: 184-191Crossref PubMed Scopus (693) Google Scholar, 5Sayre L.M. Perry G. Smith M.A. Curr. Opin. Chem. Biol. 1999; 3: 220-225Crossref PubMed Scopus (206) Google Scholar). These metals are vital for life because of their high redox activity and have been utilized in a number of enzymatic pathways, including cellular respiration. However, if the redox reactivity of copper and iron is not strictly regulated, this can result in the generation of toxic reactive oxygen intermediates (2Smith M.A. Richey Harris P.L. Sayre L.M. Beckman J.S. Perry G. J. Neurosci. 1997; 17: 2653-2657Crossref PubMed Google Scholar). The potential for oxidative damage from reactive oxygen intermediates in the aging brain is further enhanced by the high oxygen consumption and relatively low antioxidant levels in brain tissue. To prevent transition metal-mediated oxidative stress, cells have evolved elaborate systems for copper storage and transport that deliver copper and iron to metalloenzymes and proteins. A number of studies have implicated cell surface metalloreductases in the reduction of Cu(II) to Cu(I), which is the form of the metal ion that is delivered to the cytoplasm of eukaryotic cells via copper transporters (6Puig S. Thiele D.J. Curr. Opin. Chem. Biol. 2002; 6: 171-180Crossref PubMed Scopus (570) Google Scholar). To avoid Cu(I) redox chemistry inside the cell, Cu(I) ions are escorted by specific cytosolic metalloproteins such as the copper chaperones that are involved in intracellular copper trafficking to Wilson's disease copper ATPase and the copper/zinc superoxide dismutase (7Waggoner D.J. Bartnikas T.B. Gitlin J.D. Neurobiol. Dis. 1999; 6: 221-230Crossref PubMed Scopus (754) Google Scholar). This results in unbound copper being essentially absent in the intracellular environment (8Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Cr
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