Spectrin superfamily proteins play important roles in cells by interlinking numerous cytoskeletal parts and bridging the cytoskeleton to each the cell membrane and the nucleus. Characterised by the spectrin repeat (SR) area, this superfamily encompasses a distinctive bundle of three antiparallel $alpha$-helices. These SRs usually seem as tandem repeats linked by brief segments, serving as tension-bearing structural models that assist the cytoskeleton and act as signaling hubs for quite a few proteins. Though the cooperative force-dependent unfolding of tandem spectrin repeats is well-documented, the exact molecular mechanisms stay unclear. On this examine, we used the paradigmatic tandem SR (SR3-SR4) of $alpha$-actinin as our mannequin system. Our outcomes reveal that cooperativity arises from the salt bridges on the linker between the 2 domains. Moreover, we discovered that the salt bridge mechanically stabilizes the 2 domains, extending the lifetime of SR3-SR4 by 10 to 100 instances in comparison with particular person domains. Our full-atom MD simulations present that the linker salt bridge is a serious force-bearing level, and its disruption results in the mechanical unfolding of the domains. Lastly, combining Alphafold structural prediction and single-molecule manipulation research of different spectrin superfamily proteins, we display that linker salt bridge-mediated cooperativity and stabilization is a doubtlessly conserved molecular mechanism governing the mechanical responses of SRs in spectrin superfamily proteins.
