Molecular dynamics simulations show that αIIb integrin with a missing C490-C545 disulfide bond requires higher forces to transition from bent to open conformational states. (A) Ablation of the αIIb C490-C545 disulfide bond in BHK cells reduces integrin activation by manganese. The bars and errors are mean ± standard deviation of 3 independent experiments with 2 biological replicates each. (B) Molecular dynamics simulations were performed on the Thigh, Genu, and Calf-1 domain of αIIb integrin using the crystal structure of “bent” αIIbβ3 integrin was used to model the effect of the intact or missing disulfide bond (PDB identifier 3fcs).⁷ Conformational distribution of the αIIbβ3 integrin with an intact (top) and missing (bottom) C490-C545 disulfide bond, projected along the first 2 principal components (PC1 and PC2, respectively), supporting the notion that the redox state of the bond has significant structural implications. Conformational density (normalized, arbitrary units) is indicated from low (dark blue) to high (cyan). (C) Difference network of averaged pairwise-forces⁴⁸ (stick representation), measured between the integrin in both redox states. Force magnitude is indicated by stick color, from blue to green; only forces >25 kJ/mol nm are presented. Significant differences in the force networks are observed in the Genu domain, between the Thigh and Calf-1 domains, despite being distant from the missing disulfide, indicating the propagation of subtle mechanical forces throughout the protein structure. The C490-C545 disulfide is shown in stick representation, and a calcium ion is represented as an orange sphere. (D) Potential of mean force calculations suggest that the closed form of the integrin is disfavored when the C490-C545 bond is missing, reducing cycling efficiency. Representative structures from umbrella sampling are overlaid at 60, 100, and 140 degrees . **P < .01. MFI, mean fluorescence intensity.