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PDBsum entry 1soa

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Protein binding PDB id
1soa
Jmol
Contents
Protein chain
187 a.a. *
Waters ×268
* Residue conservation analysis

References listed in PDB file
Key reference
Title The parkinson'S disease protein dj-1 is neuroprotective due to cysteine-Sulfinic acid-Driven mitochondrial localization.
Authors R.M.Canet-Avilés, M.A.Wilson, D.W.Miller, R.Ahmad, C.Mclendon, S.Bandyopadhyay, M.J.Baptista, D.Ringe, G.A.Petsko, M.R.Cookson.
Ref. Proc Natl Acad Sci U S A, 2004, 101, 9103-9108. [DOI no: 10.1073/pnas.0402959101]
PubMed id 15181200
Abstract
Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1. WT or C53A DJ-1 was readily oxidized in cultured cells, generating a pI 5.8 isoform, but an artificial C106A mutant was not. We observed a cysteine-sulfinic acid at C106 in crystalline DJ-1 but no modification of C53 or C46. Oxidation of DJ-1 was promoted by the crystallization procedure. In addition, oxidation-induced mitochondrial relocalization of DJ-1 and protection against cell death were abrogated in C106A but not C53A or C46A. We suggest that DJ-1 protects against neuronal death, and that this is signaled by acidification of the key cysteine residue, C106.
Figure 1.
Fig. 1. Generation of stable dimeric cysteine mutants of human DJ-1. (a) The crystal structure of human DJ-1 with cysteine residues highlighted. Two cysteine residues, C46 and C53, whose sidechains are shown in red, are present in a -turn close to the dimer interface. A third cysteine, C106, is within the center of the dimer. (b) Transient transfections of V5-tagged cysteine mutants into M17 cells. Because some variants, notably C46A, were unstable, samples in lanes 7-11 were treated with 5 µM MG132 for 24 h. Western blots were probed with anti-DJ-1 (Upper) to visualize both transfected DJ-1 (arrows) and endogenous DJ-1 (arrowhead). The blot was reprobed with -actin as a loading control (Lower, open arrowhead). Markers on the right of the blot are in kilodaltons. (c) To assess whether these variants form dimers, transfected cells were treated with the crosslinker disuccinimidyl suberate before extraction and Western blotting for V5 (Upper) or DJ-1 (Lower). Monomeric V5-tagged protein is indicated with an arrow, and endogenous DJ-1 in the cells indicated by an arrowhead. The V5-tagged proteins formed dimers with either endogenous or transfected DJ-1, indicated by lines on the left of the blot. Blots are representative of at least duplicate experiments. To confirm these results, we coimmunoprecipitated V5-tagged DJ-1 with endogenous protein (d). (Upper) Lysates of transfected cells probed with V5 or DJ-1 to demonstrate input levels of proteins. (Lower) Lysates immunoprecipitated withV5 antibody and blotted with DJ-1 antibody to show interaction of endogenous DJ-1 with the tagged proteins.
Figure 4.
Fig. 4. DJ-1 localizes to the outer mitochondrial membrane after oxidative stress. (a) M17 cells were transfected with V5-tagged DJ-1 variants as indicated and stained for DJ-1 (V5, green) and mitotracker (red). After exposure to PQ2+ (Lower), DJ-1 colocalized with mitochondria much more extensively than under control conditions (Upper), as evidenced by the yellow color where staining overlaps. The C106A and C106D variants did not relocalize to mitochondria, whereas C53A and C46A did. This finding is quantified in b by counting the proportion of V5-positive cells that showed strong colocalization with mitotracker. Representative data are shown from at least three experiments for each construct, performed in duplicate. (c) DJ-1 is not imported into mitochondria. Cells transfected with WT DJ-1 were exposed to PQ2+ and mitochondrial fractions prepared. Duplicate samples were either untreated (-, lanes 1 and 2) or subjected to limited proteolysis with trypsin (+, lanes 3 and 4) and blotted for V5 (Top), the outer mitochondrial membrane protein VDAC1 (Middle), or the inner mitochondrial membrane protein Tim23 (Bottom).
Secondary reference #1
Title The 1.1-A resolution crystal structure of dj-1, The protein mutated in autosomal recessive early onset parkinson'S disease.
Authors M.A.Wilson, J.L.Collins, Y.Hod, D.Ringe, G.A.Petsko.
Ref. Proc Natl Acad Sci U S A, 2003, 100, 9256-9261. [DOI no: 10.1073/pnas.1133288100]
PubMed id 12855764
Full text Abstract
Figure 1.
Fig. 1. (a and b) Two views of the DJ-1 crystallographic dimer. Monomer A is purple and monomer B is green. The view in b is rotated by 90° with respect to the view in a. In both views, Cys-106 is yellow and Leu-166, which is mutated to proline in PARK7 familial PD, is red. In b, the unusual coaxial arrangement of the two C-terminal -helices (G and H) at the dimer interface is highlighted. The figure was made with POVSCRIPT+ (50).
Figure 4.
Fig. 4. Residues near the nucleophile elbow region of DJ-1 do not comprise a catalytic triad. (a) A ribbon diagram of the DJ-1 monomer is shown, with residues Glu-18, Cys-106, and His-126 in yellow. Leu-166, which is mutated to proline in PARK7 familial PD, is red. (b) A closer view of the nucleophile elbow region with 2F[o] - F[c] electron density contoured at 1.0 . Although the identity of the residues is correct for a catalytic triad, their conformation prohibits proton shuttling by the canonical mechanism of a serine/cysteine protease or a glutamine amidotransferase. The figure was made with POVSCRIPT+ (50).
PROCHECK
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