Structural Studies of the Oligomerization Process of Human Cystatin C Variants

Human cystatin C (HCC), an inhibitor of cysteine proteases, is also involved in amyloidogenic processes within the human body. In the crystal structure, as in solution, wild-type (WT) cystatin C dimerizes via a domain swapping mechanism [1,2]. The same process has also been shown to be important for protein oligomerization [2]. Aggregation of HCC results in a heterogeneous mixture of species, both oligomeric and fibrillar, with varying shapes, sizes and molecular weights.The goal of our study was to characterize the assembly process of potentially neurotoxic oligomers of WT and variant HCC in solution. The oligomerization processes of several point mutation variants of HCC (V57N, V57P, V57D, V57G and L68V), with specific dimerization propensities [3], were compared to the WT protein. Prepared oligomers were purified by size-exclusion chromatography and using TEM and AFM studies, we observed characteristic donut-like structures along with fibrils and we determined the size distribution of cystatin C oligomers obtained from the WT and variant forms. Independently, the oligomerization process was analyzed by fluorescence spectroscopy and visualized by native electrophoresis. Additionally, induced secondary structure changes were characterised by circular dichroism (CD) and infrared (FTIR) spectroscopies. Finally, the structural studies were supplemented with the modelling of the larger HCC oligomers using molecular dynamics simulations, allowing the identification of the most stable oligomeric forms of HCC to be determined.This research project was financed by the National Science Centre (Poland), decision no. 2012/06/M/ST4/00036.[1] Janowski R, Kozak M, Jankowska E et al. (2001). Nature Struct. Mol. Biol. 8, 316.[2] Wahlbom M, Wang X, Lindstrom V, et al. (2007). J. Biol. Chem. 282, 18318.[3] Szymanska A, Jankowska E, Orlikowska M, et al. (2012) Front. Mol. Neurosci., 5, 82