Abstract
TDP-43 is an RNA/DNA-binding protein, playing multiple roles in transcription repression, translation regulation, mRNA splicing and mRNA transport. However, TDP-43 is cleaved into 25-kD and 35-kD C-terminal fragments (TDP-25 and TDP-35) and aggregated in neuronal cells linking to various neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). About 50 mutations in TDP-43 have been identified in ALS patients, but it is unclear why these mutations are linked to protein aggregation and ALS. Here we purified eight ALS-linked TDP-43 mutants and found that two of the mutants, D169G and M311V, had increased thermal stability as measured by circular dichroism and differential scanning fluorimetry. To decipher the structural basis for the increased thermal stability, we determined the crystal structure of the TDP-43 RRM1 domain with D169G mutation (RRM1-D169G) in complex with a single-stranded DNA. We found that a β-turn (Turn6) in RRM1 is slightly shifted due to the loss of a hydrogen bond between G169 and T115 in RRM1-D169G. The molecular dynamic simulation further showed that the D169G increases the hydrophobic interactions in the core of the RRM1 domain, thus enhancing protein stability. Moreover, comparing to the wild type, TDP-43 with D169G mutation was cleaved more efficiently than the wild-type by caspase 3 to yield TDP-35 that may initiate protein accumulation. Consistently, expression of TDP-43 with D169G mutation in Neuro2a cells produced higher level of TDP-35 than that of wild-type protein. Taken together these results provide the structural basis for the increased stability of the TDP-43 D169G mutant, and demonstrate that D169G is more susceptible to proteolytic cleavage by caspase 3 into the stable pathogenic C-terminal 35-kD fragments that can promote protein accumulation and aggregation. Our results suggest that protein stability is an important factor for regulating TDP-43 accumulation and aggregation. Modulation of TDP-43 protein stability and caspase digesting rate could offer an avenue for prevention and treatment of neurodegenerative diseases related to TDP-43 proteinopathy.