|Title||Structure-function studies of the Parkinsonism-linked protein DJ-1|
DJ-1 is a multifunctional protein linked to familial Parkinson’s disease. DJ-1 has been suggested to exert its cytoprotective function, in part, by acting as a copper carrier that can sequester the reactive metal and/or provide the copper cofactor for the activation of the Cu-Zn superoxide dismutase (SOD1). Using absorption spectroscopy and mass spectrometry, we found that DJ-1 binds one Cu(I) ion per DJ-1 homodimer. The structure of Cu(I)-bound DJ-1 reveals a new biscysteinate metal binding motif formed by juxtaposed Cys-53 at the homodimer interface. We calculated a subfemtomolar dissociation constant (Kd = 6.41 x 10-16 M) for Cu(I) that supports the physiological intracellular retention of the metal. Cu(I)-bound DJ-1 was not capable of interacting and activating SOD1 in vitro. We posit that DJ-1 sequester copper to protect against metal-induced cytotoxicity. Our results illuminate the molecular basis on how disease-linked mutations that impairs homodimerisation could disrupt the metal binding site.
In the second part of this dissertation, we sought to determine the impact of a Parkinsonism-linked A107P mutation on DJ-1 structure and glyoxalase activity. The A107P variant abrogates the ability of DJ-1 to protect against glyoxal-induced cytotoxicity and carboxymethyllysine protein modification. A crystal structure of DJ-1 C106S variant with glycerol and sulphate bound in the active site suggests that Ala-107 is critical for the stabilization of the transition state of the nucleophilic addition step. In our hands, the protein levels of DJ-1 A107P mutant in SH-SH5Y cells were ostensibly similar to the wild-type level but reduced levels were found in HEK 293E and MEF cells. Using CD and NMR spectroscopy, we found that the structural defect caused by the mutation extends beyond the active site. The A107P mutation resulted in a remarkable misfolding of the protein providing a basis for the reduced intracellular protein level and the abrogation of enzymatic activity.