Investigation of the impacts of Parkinson's-disease-associated mutations (193M and S18Y) on the structure of human ubiquitincarboxyl-terminal hydrolase L1

Abstract

Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a protein of 223 amino acids, is a member of deubiquitinating enzymes and it is one of the most abundant proteins in the brain. Although the in vivo functions of UCH-L1 are still unclear, its abundance and specificity for neurons indicate that it may serve an important role in neuronal cell function or dysfunction. Indeed, an isoleucine 93 to methionine amino acid mutation (I93M) in UCH-L1 was identified to be linked to an autosomal dominant form of Parkinson’s disease, while the serine 18 to tyrosine amino acid mutation (S18Y) in UCH-L1 is linked to a decreased susceptibility to Parkinson’s disease.

To investigate the effects of these mutations on the structure of human UCH-L1, the mutant proteins have been successfully over-expressed, biophysically characterized and compared with the wild-type UCH-L1 using circular dichroism and NMR spectroscopy. While the data from circular dichroism and NMR chemical shift perturbation analysis suggested that the S18Y point mutation only slightly perturbs the global structure, the effect of the I93M point mutation was found to be more profound. In particular, the structural perturbations caused by I93M substitution are not only observed near the site of mutation, but are also found at more distant sites. These structural perturbations may be significant for the function of UCH-L1 and explain the reduced hydrolase activity (~55 % of wild-type) observed in UCH-L1-I93M, as the geometry of the catalytic triad (C90, H161 and D176) is likely to be distorted by this substitution.

To provide further insights into the effect of serine 18 to tyrosine (S18Y) mutation on the structure and function of UCH-L1, the three-dimensional solution structure of UCH-L1-S18Y was determined by NMR spectroscopy. The solution structure of UCH-L1-S18Y reveals a monomer with a typical fold of papain-like cysteine proteases and consists of a six-membered antiparallel β-sheet surrounded by eight α-helices. Although the global structure is very similar to the crystal structure of wild-type UCH-L1, both the altered hydrogen bond network and the surface charge distributions have demonstrated that the S18Y substitution could lead to profound structural changes. In particular, the analysis of the difference in the dimeric interfaces of the wild-type and the S18Y mutant showed that the serine to tyrosine mutation can significantly affect the distribution of the surface-exposed residues involved in the dimeric interface. It is thought that such observed differences might weaken the stability of the UCH-L1 dimer and hence may explain the reduced dimerization-dependent ligase activity of UCH-L1-S18Y in comparison to the wild-type UCH-L1.