title: Impact of autosomal recessive juvenile Parkinson's disease mutations on the structure and interactions of the parkin ubiquitin-like domain.
abstract: Autosomal recessive juvenile parkinsonism (ARJP) is an early onset familial form of Parkinson's disease. Approximately 50% of all ARJP cases are attributed to mutations in the gene park2, coding for the protein parkin. Parkin is a multidomain E3 ubiquitin ligase with six distinct domains including an N-terminal ubiquitin-like (Ubl) domain. In this work we examined the structure, stability, and interactions of the parkin Ubl domain containing most ARJP causative mutations. Using NMR spectroscopy we show that the Ubl domain proteins containing the ARJP substitutions G12R, D18N, K32T, R33Q, P37L, and K48A retained a similar three-dimensional fold as the Ubl domain, while at least one other (V15M) had altered packing. Four substitutions (A31D, R42P, A46P, and V56E) result in poor folding of the domain, while one protein (T55I) showed evidence of heterogeneity and aggregation. Further, of the substitutions that maintained their three-dimensional fold, we found that four of these (V15M, K32T, R33Q, and P37L) lead to impaired function due to decreased ability to interact with the 19S regulatory subunit S5a. Three substitutions (G12R, D18N, and Q34R) with an uncertain role in the disease did not alter the three-dimensional fold or S5a interaction. This work provides the first extensive characterization of the structural effects of causative mutations within the ubiquitin-like domain in ARJP.
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1.
authors.forename: Susan S
authors.initials: SS
authors.lastname: Safadi
authors.forename: Kathryn R
authors.initials: KR
authors.lastname: Barber
authors.forename: Gary S
authors.initials: GS
authors.lastname: Shaw
mesh_terms: D019943:Amino Acid Substitution; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D020734:Parkinsonian Disorders / Q000235:genetics* / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011489:Protein Denaturation; D054730:Protein Interaction Domains and Motifs; D058849:Protein Refolding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D012335:RNA, Ribosomal / Q000378:metabolism; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; C498506:PSMD4 protein, human; D012335:RNA, Ribosomal; C097639:RNA, ribosomal, 19S; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D046988:Proteasome Endopeptidase Complex