Transient sampling of aggregation‐prone conformations causes pathogenic instability of a parkinsonian mutant of DJ‐1 at physiological temperature

Abstract

Various missense mutations in the cytoprotective protein DJ-1 cause rare forms of inherited parkinsonism. One mutation, M26I, diminishes DJ-1 protein levels in the cell but does not result in large changes in the three-dimensional structure or thermal stability of the protein. Therefore, the molecular defect that results in loss of M26I DJ-1 protective function is unclear. Using NMR spectroscopy near physiological temperature, we found that the picosecond-nanosecond dynamics of wild-type and M26I DJ-1 are similar. In contrast, elevated amide hydrogen/deuterium exchange rates indicate that M26I DJ-1 is more flexible than the wild-type protein on longer timescales and that hydrophobic regions of M26I DJ-1 are transiently exposed to solvent. Tryptophan fluorescence spectroscopy and thiol crosslinking analyzed by mass spectrometry also demonstrate that M26I DJ-1 samples conformations that differ from the wild-type protein at 378C. These transiently sampled conformations are unstable and cause M26I DJ-1 to aggregate in vitro at physiological temperature but not at lower temperatures. M26I DJ-1 aggregation is correlated with pathogenicity, as the structurally similar but non-pathogenic M26L mutation does not aggregate at 378C. The onset of dynamically driven M26I DJ-1 instability at physiological temperature resolves conflicting literature reports about the behavior of this disease-associated mutant and illustrates the pitfalls of characterizing proteins exclusively at room temperature or below, as key aspects of their behavior may not be apparent.

15N CEST data and traditional model-free analysis capture fast internal dynamics of DJ-1

“…The fast protein dynamics of reduced DJ-1 have been previously determined using traditional spin relaxation derived R 1 and R 2 values and 1 H- 15 N NOE data [16]. DJ-1 was found to be fairly rigid in solution at 35˚C, with most order parameters ( S 2 ) approaching the maximum theoretical limit ( S 2 = 1).…”

“…The calculated RMSDs values (Table 3) comparing the CEST and traditional R 1 and R 2 values are within the observed standard deviations for the data sets (Tables 1 and 2). An NOE RMSD of 0.13 was obtained when we compared our original 1 H- 15 N NOE data [16] with an NOE data set recollected with the CEST experiments. Again, the NOE RMSD is within the observed experimental error and standard deviations (Tables 1 and 2).…”

“…The R 1 , R 2 , NOE, and S 2 errors reported in Tables 1 and 2 were calculated as the standard error of the mean. Heteronuclear NOE experiments were performed as previously described [16] and processed in NMRPipe. FAST-ModelFree analysis was done identical to our previous study to ensure a proper comparison of the methods [16].…”

The effect of cysteine oxidation on DJ-1 cytoprotective function in human alveolar type II cells

“…Two-dimensional (2D) 1 H- 15 N heteronuclear single quantum coherence (HSQC) spectra were collected at 37 °C as previously described 42,43 from uniformly 15 N labeled DJ-1 that was either reduced or overoxidized to Cys106-SO 3 − The 2D 1 H- 15 N HSQC spectra were collected on a 700 MHz Bruker Avance III spectrometer equipped with a 5 mm quadruple resonance QCI-P cryoprobe ( 1 H, 13 C, 15 N, and 31 P) with z -axis gradients. Order parameters ( S 2 ) were calculated from chemical exchange saturation transfer (CEST)-derived R 1 and R 2 relaxation rates and heteronuclear NOE values using FAST-Modelfree 44 as previously described 42 .…”

Regulation of Signal Transduction by DJ-1

“…L166P and M26I mutations affect DJ-1 stability (although with mixed results for M26I), and its ability to form homodimers, and result in reduced cellular DJ-1 levels (Moore et al 2003; Takahashi-Niki et al 2004; Milkovic et al 2015). E64D mutant DJ-1 may retain the ability to form homodimers, but this mutation may potentiate DJ-1 aggresome formation (Repici et al 2013).…”