A Conserved Flexible N-Terminal Domain Tunes the Calcium Sensitivity of Sorcin by Stabilizing Its Active Conformation

Sorcin is a penta-EF-hand Ca ²⁺ -binding protein that acts as a Ca ²⁺ sensor and regulator of Ca ²⁺ homeostasis. Although the structure and Ca ²⁺ -dependent activation of Sorcin is well characterized, the function of its flexible N-terminal domain (NTD) remains unclear. We combined sequence analysis, Ca ²⁺ -induced aggregation assays, multidimensional NMR spectroscopy, and long-timescale molecular dynamics (MD) simulations to define the NTD’s role in Sorcin activation. Sequence comparisons showed that the NTD is conserved across vertebrates despite its intrinsic disorder, indicating functional importance. NTD deletion markedly reduced Ca ²⁺ responsiveness. Relative to full-length Sorcin, the construct with NTD truncation required more than twofold higher Ca ²⁺ concentrations and over tenfold higher protein concentrations to initiate aggregation, while aggregation kinetics slowed by nearly three orders of magnitude. Temperature-dependent measurements yielded an apparent activation energy of 36.7 kJ·mol ^-1 , consistent with aggregation driven by Ca ²⁺ -induced conformational activation rather than denaturation. NMR chemical shift perturbations localized the effects of NTD removal to the EF-hand Ca ²⁺ -binding loops and adjacent D-helix. Apo-state MD simulations revealed transient intra- and intermolecular NTD-SCBD contacts that explain some perturbations and support direct and allosteric regulation. Ca ²⁺ -bound simulations further showed that SCBD departs more readily from the crystallographic active conformation than full-length Sorcin, indicating that the NTD stabilizes the active state through dynamic contacts. Together, these findings establish the NTD as a critical regulator that enhances Ca ²⁺ responsiveness by shifting Sorcin’s conformational equilibrium toward the active state and suggest that flexible N-terminal extensions regulate signaling within the penta-EF-hand protein family.