Triple Calcium Binding Stoichiometry in the Monoclinic Crystal Form of Protracted Insulin

Insulin is stored in pancreatic β ‐cell granules as Zn ²⁺ and Ca ²⁺ ‐stabilized hexamers, yet the structural impact of divalent cations, particularly the role of Ca ²⁺ , remains incompletely understood. Here, the first crystal structures of insulin hexamers coordinated by three Ca ²⁺ cations are reported, determined in monoclinic space group P12 ₁ 1 under near‐physiological (293 K) and cryogenic (100 K) temperatures. Unlike classical rhombohedral crystal forms, these structures suggest direct evidence of calcium binding without relying on symmetry averaging. Structural and molecular dynamics analyses reveal that 1) three Ca ²⁺ cations occupy the GluB13 (13th glutamic acid in chain B)‐centered cavity, forming bridging interactions between adjacent monomers via carboxylate coordination, 2) calcium binding preserves the canonical Zn ²⁺ –HisB10 coordination and maintains a structured hydrogen‐bond network involving GluB13, HisB10, and water molecules, 3) AsnB3 residues of each monomer remain flexible yet spatially clustered, suggesting that Ca ²⁺ supports electrostatic interactions critical for allosteric R ₆ ‐state stabilization, and 4) compared to calcium‐free forms, these structures show enhanced oligomeric coherence, implying the maintenance of the R ₆ hexamer stability. Combined with computational analyses, these findings uncover a native‐like, Ca ²⁺ ‐driven assembly mechanism that enhances hexamer cavity stability through inter‐subunit coordination, offering a biomimetic strategy for the rational design of long‐acting insulin analogs.