BPS2026 – Highly cooperative interaction between cytoskeletal adaptor KANK1 and hub protein LC8 forms dynamic hinged rods, suggesting a structural role for LC8 in cytoskeletal assembly

KANK1 is a cytoskeletal regulator localized to the cell cortex, where it binds to both focal adhesions and cortical microtubule-stabilizing complexes. LC8 is a small dimeric hub that dimerizes many clients through binding, in some cases multivalently, at short linear motifs. While the exact function of each multivalent LC8 client is unique, these interactions play a structural role, rigidifying a disordered region of the client protein. Using in-cell and in vitro assays, we identify KANK1 as a new LC8 interaction partner, which drives LC8 localization to the cell cortex. Because the LC8 recognition motifs on KANK1 are all individually weak and non-canonical in nature, our prediction algorithm LC8Pred only produced low-confidence predictions and was insufficient to find all sites inferred from experiments. Therefore, we employed our recently developed method, which capitalizes on built-in confidence metrics of AlphaFold predictions to complement LC8Pred. This method located 15 recognition motifs in the central 660 amino acid disordered linker of KANK1. Isothermal titration calorimetry, analytical ultracentrifugation, and multi-angle light scattering support AlphaFold predictions and demonstrate that KANK1∖LC8 binding is highly cooperative, with an overall binding affinity at least two orders of magnitude greater than the affinity of each individual motif. Consistent with multivalent AlphaFold predictions, negative-stain electron microscopy resolves 15 LC8 dimers in a row with varying dynamics around a hinged-rod-like structure. We thus propose a new structural role for LC8, with multivalent cooperativity building a rod, elongating the long, disordered linker, and enabling KANK1 to reach across the gap between the cell membrane and the ends of microtubules.