Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by
keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these
proteins cause
epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented
plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of
plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on
enhanced green fluorescent protein-tagged
proteins (FluoBACE). Our results demonstrate that all four
plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The
plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric
keratins or IF assembly intermediates. These findings indicate a multimodular association of
plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in
plectin, K5, or K14 genes.