Spectrin superfamily proteins play essential roles in cells by interlinking various cytoskeletal components and bridging the cytoskeleton to both the cell membrane and the nucleus. Characterized by the spectrin repeat (SR) domain, this superfamily features a unique bundle of three antiparallel $\alpha$-helices. These SRs often appear as tandem repeats linked by short segments, serving as tension-bearing structural units that support the cytoskeleton and act as signaling hubs for numerous proteins. Although the cooperative force-dependent unfolding of tandem spectrin repeats is well-documented, the precise molecular mechanisms remain unclear. In this study, we used the paradigmatic tandem SR (SR3-SR4) of $\alpha$-actinin as our model system. Our results reveal that cooperativity arises from the salt bridges on the linker between the two domains. Additionally, we found that the salt bridge mechanically stabilizes the two domains, extending the lifetime of SR3-SR4 by 10 to 100 times compared to individual domains. Our full-atom MD simulations show that the linker salt bridge is a major force-bearing point, and its disruption leads to the mechanical unfolding of the domains. Finally, combining Alphafold structural prediction and single-molecule manipulation studies of other spectrin superfamily proteins, we demonstrate that linker salt bridge-mediated cooperativity and stabilization is a potentially conserved molecular mechanism governing the mechanical responses of SRs in spectrin superfamily proteins.
