A disease state mutation unfolds the parkin ubiquitin-like domain.
E3 ubiquitin ligases are essential enzymes in the ubiquitination pathway responsible for the recognition of specific E2 conjugating enzymes and for transferring ubiquitin to a substrate targeted for degradation. In autosomal recessive juvenile Parkinson's disease, an early onset form of Parkinson's disease, point mutations in the E3 ligase parkin are one of the most commonly observed traits. Parkin is a multidomain E3 ligase that contains an N-terminal ubiquitin-like domain that interacts with, and effects the ubiquitination of, substrates such as cyclin E, p38 and synphilin. In this work we have examined the folding and structure of the parkin ubiquitin-like domain (Ubld) and of the protein with two causative disease mutations (K48A and R42P). Parallel experiments with the protein ubiquitin were done in order to determine if the same mutations were detrimental to the ubiquitin structure and stability. Despite similar folds between the parkin Ubld and ubiquitin, urea unfolding experiments show that the parkin Ubld is surprisingly approximately 10.6 kJ/mol less stable than ubiquitin. The K48A mutation had little effect on the stability of the parkin Ubld or ubiquitin indicating that this mutation contributes to defective protein-protein interactions. In contrast, the single point mutation R42P in parkin's Ubld caused poor expression and degradation of the protein. To avoid these problems, a GB1-Ubld fusion protein was characterized by NMR spectroscopy to show that the R42P mutation causes the complete unfolding of the parkin Ubld. This observation provides a rationale for the more rapid degradation of parkin carrying the R42P mutation in vivo, and its inability to interact with some substrate proteins. Our work provides the first structural and folding insight into the effects of causative mutations within the ubiquitin-like domain in autosomal recessive juvenile Parkinson's disease.
Safadi
Susan S
SS
Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1.
Shaw
Gary S
GS
D002942:Circular Dichroism; D006801:Humans; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics*
D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein