Optimal Mutant Model of Human S100A3 Protein Citrullinated at Arg51 by Peptidylarginine Deiminase Type III and Its...

S100A3 protein, a member of the EF-hand-type Ca²⁺-binding S100 protein family, undergoes a Ca²⁺-/Zn²⁺-induced structural change to a tetrameric state upon specific citrullination of R51 in human hair cuticular cells. To elucidate the underlying mechanism, we prepared recombinant mutant S100A3 proteins, including R51A, R51C, R51E, R51K, and R51Q, as potential models of post-translationally modified S100A3 and evaluated their biophysical and biochemical properties relative to wild-type (WT) S100A3 and WT citrullinated in vitro. Size exclusion chromatography (SEC) showed that R51Q formed a tetramer in the presence of Ca²⁺, while Ca²⁺ titration monitored by Trp fluorescence indicated that R51Q had Ca²⁺-binding properties similar to those of citrullinated S1003A. We therefore concluded that R51Q is the optimal mutant model of post-translationally modified S100A3. We compared the solution structure of WT S100A3 and the R51Q mutant in the absence and presence of Ca²⁺ and Zn²⁺ by SEC-small-angle X-ray scattering. The radius of gyration of R51Q in the metal-free state was almost the same as that of WT; however, it increased by ∼1.5-fold in the presence of Ca²⁺/Zn²⁺, indicating a large expansion in molecular size. By contrast, addition of Ca²⁺/Zn²⁺ to WT led to nonspecific aggregation in SEC analysis and dynamic light scattering, suggesting that citrullination of S100A3 is essential for stabilization of the Ca²⁺-/Zn²⁺-bound state. These findings will lead to the further development of structural analyses for the Ca²⁺-/Zn²⁺-bound S100A3.