Refined Crystal Structures of Human Ca2+/Zn2+-Binding S100A3 Protein Characterized by Two Disulfide Bridges

S100A3, a member of the EF-hand-type Ca2+-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn2+ affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca2+/Zn2+ supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A + C68A) abolished the Ca2+ binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca2+ affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A + C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15−1.40 Å resolution shows that SS1 renders the C-terminal classical Ca2+-binding loop flexible, which are essential for its Ca2+ binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn2+ by (Cys)3His residues that is compatible with SS2 formation in S100A3.