Crystal Structure of Human DJ-1, a Protein Associated with Early Onset Parkinson's Disease

We report the crystal structure at 1.8-Å resolution of human DJ-1, which has been linked to early onset Parkinson's disease. The monomer of DJ-1 contains the α/β-fold that is conserved among members of the DJ-1/ThiJ/PfpI superfamily. However, the structure also contains an extra helix at the C terminus, which mediates a novel mode of dimerization for the DJ-1 proteins. A putative active site has been identified near the dimer interface, and the residues Cys-106, His-126, and Glu-18 may play important roles in the catalysis by this protein. Studies with the disease-causing L166P mutant suggest that the mutation has disrupted the C-terminal region and the dimerization of the protein. The DJ-1 proteins may function only as dimers. The Lys to Arg mutation at residue 130, the site of sumoylation of DJ-1, has minimal impact on the structure of the protein. We report the crystal structure at 1.8-Å resolution of human DJ-1, which has been linked to early onset Parkinson's disease. The monomer of DJ-1 contains the α/β-fold that is conserved among members of the DJ-1/ThiJ/PfpI superfamily. However, the structure also contains an extra helix at the C terminus, which mediates a novel mode of dimerization for the DJ-1 proteins. A putative active site has been identified near the dimer interface, and the residues Cys-106, His-126, and Glu-18 may play important roles in the catalysis by this protein. Studies with the disease-causing L166P mutant suggest that the mutation has disrupted the C-terminal region and the dimerization of the protein. The DJ-1 proteins may function only as dimers. The Lys to Arg mutation at residue 130, the site of sumoylation of DJ-1, has minimal impact on the structure of the protein. Parkinson's disease (PD) 1The abbreviations used are: PD, Parkinson's disease; PIASxα, protein inhibitor of activated STAT; STAT, signal transducers and activators of transcription; DJBP, DJ-1-binding protein; GAT, glutamine amidotransferase; r.m.s., root mean square; HPII, hydroperoxidase II. is a common, progressive neurodegenerative disorder affecting roughly 1% of the population at the age of 65 (1Dawson T.M. Dawson V.L. J. Clin. Invest. 2003; 111: 145-151Crossref PubMed Scopus (206) Google Scholar). Clinically, PD generally presents with brady-kinesia, resting tremor, muscular rigidity, and postural instability. PD is a heterogeneous disease, and the majority of the cases appear to have sporadic origins. At the same time, the disorder can also be associated with specific genetic defects, especially for cases of familial PD (2Gasser T. J. Neurol. 2001; 248: 833-840Crossref PubMed Scopus (128) Google Scholar, 3Giasson B.I. Lee V.M.-Y. Neuron. 2001; 31: 885-888Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4Cookson M.R. Neuron. 2003; 37: 7-10Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Ten different genetic loci have been linked with familial PD, and the genes responsible for PD at these loci include the presynaptic protein α-synuclein (5Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6734) Google Scholar), parkin (6Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4231) Google Scholar), and ubiquitin carboxyl-terminal hydrolase L1 (7Liu Y. Fallon L. Lashuel H.A. Liu Z. Lansbury Jr., P.T. Cell. 2002; 111: 209-218Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar). Most recently, it was discovered that mutations in the DJ-1 gene are linked with autosomal recessive early onset familial PD (8Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2287) Google Scholar). Two types of disruptions of this gene have been identified in PD patients. One is a deletion of several of its exons, which abolishes the production of the DJ-1 protein. The other disruption is a single point mutation, giving rise to the L166P mutant at the protein level. This mutant has a different cytoplasmic distribution and is believed to be functionally inactive. Therefore, loss of the normal function of DJ-1 appears to contribute to PD. The exact biological function of the DJ-1 protein is currently unknown. It may play a role in the oxidative stress response, and this function could be important in preventing the onset of PD (8Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2287) Google Scholar). Both α-synuclein and parkin participate in oxidative stress responses as well (9Hashimoto M. Hsu L.J. Rockenstein E. Takenouchi T. Mallory M. Masliah E. J. Biol. Chem. 2002; 277: 11465-11472Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 10Hyun D.-H. Lee M. Hattori N. Kubo S.-I. Mizuno Y. Halliwell B. Jenner P. J. Biol. Chem. 2002; 277: 28572-28577Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). On the other hand, DJ-1 may also be associated with several other biological processes. It was first identified as an oncogene, because it can transform NIH3T3 cells in cooperation with the ras oncogene (11Nagakubo D. Taira T. Kitaura H. Ikeda M. Tamai K. Iguchi-Ariga S.M.M. Ariga H. Biochem. Biophys. Res. Commun. 1997; 231: 509-513Crossref PubMed Scopus (672) Google Scholar). The DJ-1 protein is also involved in the fertilization process in rat and mouse (12Wagenfeld A. Gromoll J. Cooper T.G. Biochem. Biophys. Res. Commun. 1998; 251: 545-549Crossref PubMed Scopus (95) Google Scholar, 13Welch J.E. Barbee R.R. Roberts N.L. Suarez J.D. Klinefelter G.R. J. Androl. 1998; 19: 385-393PubMed Google Scholar, 14Okada M. Matsumoto K.-I. Niki T. Taira T. Iguchi-Ariga S.M.M. Ariga H. Biol. Pharm. Bull. 2002; 25: 853-856Crossref PubMed Scopus (83) Google Scholar). A significant reduction in the amount of this protein on the surface of sperm makes them unable to fertilize eggs (15Wagenfeld A. Yeung C.-H. Shivaji S. Sundareswaran V.R. Ariga H. Cooper T.G. J. Androl. 2000; 21: 954-963PubMed Google Scholar). This finding suggests that the protein may be secreted under some circumstances, which has also been observed in breast cancers (16Le Naour F. Misek D.E. Krause M.C. Deneux L. Giordano T.J. Scholl S. Hanash S.M. Clin. Cancer Res. 2001; 7: 3328-3335PubMed Google Scholar). Finally, DJ-1 was identified as the regulatory subunit of a 400-kDa RNA-binding protein complex and its presence inhibits the binding of RNA by the complex (17Hod Y. Pentyala S.N. Whyard T.C. El-Maghrabi M.R. J. Cell. Biochem. 1999; 72: 435-444Crossref PubMed Scopus (173) Google Scholar). The DJ-1 protein is a positive regulator of the androgen receptor by sequestering its negative regulators PIASxα (protein inhibitor of activated STAT) or DJBP (DJ-1-binding protein) (18Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 19Niki T. Takahashi-Niki K. Taira T. Iguchi-Ariga S.M.M. Ariga H. Mol. Cancer Res. 2003; 1: 247-261PubMed Google Scholar). The activation of the androgen receptor might be related to the effects of this protein on fertility. PIAS proteins are SUMO-1 (small ubiquitin-like modifier-1) ligases and control their target proteins by sumoylation (20Kotaja N. Karvonen U. Janne O.A. Palvimo J.J. Mol. Cell. Biol. 2002; 22: 5222-5234Crossref PubMed Scopus (355) Google Scholar). It has been reported that PIASxα can sumoylate DJ-1 on the Lys-130 residue (18Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). DJBP is almost exclusively expressed in the testis in humans. It negatively modulates the androgen receptor by recruiting histone deacetylases. DJ-1 can inhibit this recruitment, thereby leading to the activation of the androgen receptor (19Niki T. Takahashi-Niki K. Taira T. Iguchi-Ariga S.M.M. Ariga H. Mol. Cancer Res. 2003; 1: 247-261PubMed Google Scholar). The human DJ-1 protein contains 189 amino acid residues. It belongs to the DJ-1/ThiJ/PfpI superfamily of proteins, which are conserved in many different organisms (Fig. 1). The function of ThiJ is currently unknown, although it might be related to the biosynthesis of thiamin (21Mizote T. Tsuda M. Smith D.D.S. Nakayama H. Nakazawa T. Microbiology. 1999; 145: 495-501Crossref PubMed Scopus (41) Google Scholar, 22Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-H. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). PfpI is an intracellular protease that was first identified from the archaeon Pyrococcus furiosus (23Halio S.B. Blumentals I.I. Short J. PubMed Google Scholar), and it is in and The crystal structure of the related intracellular protease from Pyrococcus the presence of a residue a different with the active site at the of in a R. J. H. U. S. A. 2000; PubMed Scopus Google Scholar). A was in a of this the protein from E. K. F. U. S. A. 2003; PubMed Scopus Google Scholar). The amino acid of this only a with that of The DJ-1/ThiJ/PfpI proteins also and to of the glutamine which a 2001; PubMed Scopus Google Scholar). the residue in the active site of the PfpI is conserved among of the members of the DJ-1/ThiJ/PfpI the and residues are conserved in DJ-1 and its (Fig. 1). the of this putative active site in DJ-1 and to a for the function of this have its crystal structure at a 1.8-Å We have also the disease-causing L166P mutant in which suggests that the DJ-1 protein may function only as a and gene for human DJ-1 was the and in E. at The protein contains a at the C the protein was by and The protein was to in a and and at are residues in DJ-1, and the protein was by the The C-terminal was for the production of proteins, the was the E. The was in J. PubMed Scopus Google Scholar), and the protein was the same as for the protein. L166P and single site of human DJ-1 with the and to the of the The mutant proteins expressed in E. and the same as for the protein. of the DJ-1 and mutant proteins by the at for the protein identified by with The and The protein was at a of with from the Both and with a and and in of the the and The protein was at with and in the of with a and and in for at K. and at on an at the of The and with the Z. 1997; 276: Scopus Google Scholar). A single to 1.8-Å resolution was on a It belongs to with in the The crystal of the protein belongs to with in the A to resolution was for this The mutant crystal belongs to with a dimer in the to The are in of resolution of for of in in in a and of with the C.M. R. J. 1999; Scopus Google Scholar). to 1.8-Å resolution on the single and with the T.C. J. D. 1999; PubMed Scopus Google Scholar), which also of the residues in the The was the with the M. A. PubMed Scopus Google Scholar). The of the crystal and the mutant by the with the G. Y. L. D. 2001; PubMed Scopus Google Scholar). The structure was with the P. J. M. T. D. 1998; PubMed Scopus Google Scholar). The on the structure are in The crystal structure of human DJ-1 protein in a crystal has been at 1.8-Å resolution by the single Science. PubMed Scopus Google Scholar). The of the by C.M. R. J. 1999; Scopus Google Scholar), which the of the and of of the residues in the protein T.C. J. D. 1999; PubMed Scopus Google Scholar). The contains residues of DJ-1 with The residue of the protein and the C-terminal are in the The has with the with a of The from in and are and of the residues with the of are in of the The residue has a We have also the crystal structure at resolution of the protein in a crystal by the G. Y. L. D. 2001; PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). This crystal contains of DJ-1 in the to the of the monomer and dimer of The structure of the mutant of human DJ-1 has been at resolution by the This crystal is to of the The structure that the in are involved in with in the and these are different from for the Lys-130 in the of the DJ-1 structure of the monomer of DJ-1 has the α/β-fold with and (Fig. The of the structure contains The of this are to in the with the that the is in the DJ-1 structure with only and the and a structure and are involved in the dimerization of DJ-1 and with helix a which the conserved residue to the putative active site of DJ-1 (Fig. Most of the the of the (Fig. The only is helix at the C of It from the of the protein and only and in the monomer (Fig. in helix are conserved to be amino among the DJ-1 and ThiJ proteins (Fig. 1). residues the with and as well as the dimerization of DJ-1 The of and that DJ-1 protein as in and L. the crystal of the is a monomer of DJ-1 in the The other monomer of the dimer is related by the (Fig. The of the in the of the dimer (Fig. At of the interface, the is to the dimer that a is the dimer (Fig. At the other the with residues near the C of the other in helix (Fig. may also be important to the of the helix at the C of the crystal of the are in the among these and the dimer in the crystal that the of the are the The of of these is The helix among the in the and this helix also has in the The of the dimer is also conserved in that of the mutant with a of for of of the and mutant dimers. The DJ-1 dimer has an the A of of the surface of monomer is at this interface, residues that are conserved among DJ-1 proteins (Fig. 1). and suggest that this dimer is to be a and conserved for these proteins. a deletion mutant of DJ-1 residues to the C (Fig. is in that the helix has a role in the dimerization of DJ-1 or the C-terminal is the dimer On the other hand, the deletion mutant residues to the C of of with in of structure of DJ-1 the to that of from P. R. J. H. U. S. A. 2000; PubMed Scopus Google Scholar), a of the PfpI and the proteins amino acid The is for DJ-1 and Most of the structure are the proteins (Fig. However, the helix at the C of DJ-1 have a in suggest that of the PfpI members this because are residues DJ-1 at the C (Fig. 1). The structure of and DJ-1 are the in the and structure of their to the of DJ-1, a structure roughly was observed in the crystal of R. J. H. U. S. A. 2000; PubMed Scopus Google Scholar). This can be as a of and the of the dimer is by the and However, a mode of dimerization is with DJ-1 as it is by the extra helix in Therefore, the DJ-1 dimer is by a different of the and of the and the also contribute to the dimerization (Fig. the of the to the dimer is to DJ-1 (Fig. The dimerization of a of and DJ-1, is by the K. F. U. S. A. 2003; PubMed Scopus Google Scholar). the with the L. C. J. Mol. Biol. PubMed Scopus Google that DJ-1 also significant with the C-terminal in the hydroperoxidase J. M.J. T. J. K. 1999; PubMed Scopus Google as has been 2001; PubMed Scopus Google Scholar). The for DJ-1 and this C-terminal is The amino acid for these residues is only This in the (Fig. which for its At the C terminus, also has a helix it is in an from the helix in The exact function of this C-terminal in is It mediates the dimer in the of the and to the leading to the active site J. M.J. T. J. K. 1999; PubMed Scopus Google Scholar). This may also the of the as deletion this in the The dimerization of this in the helix at the C and is different from that in the DJ-1/ThiJ/PfpI superfamily and the has also been 2001; PubMed Scopus Google Scholar). However, the is The the structure in DJ-1 and the binding site for glutamine in is by the in the DJ-1 Therefore, it is that DJ-1 can have The of exact function of the DJ-1 protein is currently However, and with proteins can as to the roles of this protein in oxidative stress response, androgen receptor and other processes. A residue is conserved among of the members of the DJ-1/ThiJ/PfpI and the residue has been as the for the PfpI of intracellular R. J. H. U. S. A. 2000; PubMed Scopus Google Scholar). This residue is to in human DJ-1 (Fig. and is in the structure (Fig. The of this residue a with and of and which is also observed in the of and R. J. H. U. S. A. 2000; PubMed Scopus Google Scholar, K. F. U. S. A. 2003; PubMed Scopus Google Scholar). The of this residue in DJ-1 suggests that it may also have a role in the function of this this region of the structure as the putative active site of the PfpI and the the residue is of a R. J. H. U. S. A. 2000; PubMed Scopus Google Scholar, K. F. U. S. A. 2003; PubMed Scopus Google Scholar). The residue in the the residue in the of these proteins and DJ-1, the residue is (Fig. that are DJ-1 and the PfpI in this active that His-126, from the and conserved among of the DJ-1 proteins (Fig. is near the in the putative active site of DJ-1 (Fig. The residue could be the of the for The the of and is in the structure (Fig. to the of that is in the active site of A.M. R.L. 1999; 7: Full Text Full Text PDF PubMed Scopus Google Scholar). mutation of residues of DJ-1, and can the with PIASxα (18Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), for the of the However, of the residues are conserved among the DJ-1 proteins (Fig. and and appear to have important roles as Therefore, it is which of the mutations is responsible for the loss of The residues are by from other in the it is that can the with at the same the residue of the from monomer in the (Fig. R. J. H. U. S. A. 2000; PubMed Scopus Google Scholar), in the residue from an to the K. F. U. S. A. 2003; PubMed Scopus Google Scholar). of DJ-1, residues are near the The active site of the DJ-1 members may only a a residue as the it is that a is for in the On the other of the the of Glu-18 is of the in the structure of DJ-1 (Fig. This residue is conserved among of the DJ-1/ThiJ/PfpI proteins (Fig. and it is also in the of this residue is in the Therefore, it is that this residue also has an important role in the of these proteins. The of putative active site of DJ-1 is near the of the dimer with from residues in and of the other monomer (Fig. The of the dimer are in a that the active are on of the dimer (Fig. The residue in the of a surface in the structure (Fig. in this active site are generally conserved the and the (Fig. that are conserved among the DJ-1 and ThiJ proteins (Fig. 1). The helix an especially important role in this active The Glu-18 residue is near the of this and near its C contribute to the other active site of the dimer (Fig. Cys-106, is a of residues and and residues and (Fig. residues are conserved among of the members of the DJ-1/ThiJ/PfpI superfamily (Fig. 1). The of may the of the by to residues and at its (Fig. is the of helix and is to the of residue this residue with and the of the It is that and are involved in the binding of the of this protein (Fig. The of the of has been reported that the Lys-130 residue can and that this is important for DJ-1 to in (18Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). the crystal the Lys-130 residue is at the of helix from the and its is to the (Fig. structure of the mutant of DJ-1 that the mutation has impact on the structure of the protein The in the different (Fig. this is to the that the Arg residues are involved in different The is with the that the mutation the with the PIASxα protein (18Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Studies on the L166P L166P mutation of human DJ-1 was first in an PD (8Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2287) Google Scholar). This residue is conserved among the DJ-1 proteins (8Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2287) Google as well as the ThiJ proteins, although it is conserved among the PfpI (Fig. 1). on DJ-1 and the it was that the residue is in a helix and the L166P mutation may the of this helix (8Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2287) Google Scholar). that is in the of helix (Fig. that the of this residue has a role in the the extra helix at the C and the and (Fig. A of of the surface of is at this Therefore, the L166P mutation may have effects on the structure of DJ-1 of the helix and the disruption of the among and the structure of the L166P have expressed it in E. and the protein by and at of this mutant have been with the protein that the mutant is in This is with the that the L166P mutation may to the of the C-terminal of DJ-1, which also the of the suggest that the DJ-1 protein may only function as a on the DJ-1, and PfpI proteins have been a superfamily. the on DJ-1 and PfpI it is that are significant and among the members of the superfamily. DJ-1 and ThiJ proteins may be related to the PfpI proteins are of the amino acid suggests that ThiJ proteins might also have the helix at the C (Fig. to DJ-1, the PfpI proteins The of the residue in the DJ-1 and ThiJ proteins is related to the presence of this extra because it is a of the this helix and the of the protein (Fig. It is also that the ThiJ proteins the same dimer as DJ-1, in to The disease-causing effects of the L166P mutation in DJ-1 the of the of the C-terminal and that these proteins may function only as dimers. among members of this superfamily is that the active site residue the in the only in the PfpI proteins. DJ-1 and ThiJ are in an residue at this in the the residue identified from is conserved only in on the and the it is that the ThiJ proteins a residue in the active However, the Glu-18 residue is conserved in DJ-1 has been associated with many different and biological suggests that DJ-1 may an active site and could also be a to the PfpI The of the and residues are of the active site of and residues are from the residue in DJ-1, to the for the Arg residues that the residue in the of It be to DJ-1 can residues in this putative active The DJ-1 protein can also with other proteins as PIASxα, DJBP, and the RNA-binding protein It is the putative of DJ-1 is for these or a different surface can these The DJ-1 and a that is for the function of and other the that DJ-1 may also the of This could be the DJ-1 and the oxidative stress We for with at the and and for the