Phosphorylation-dependent conformational change in the Keratin 19 head domain: Implications for filament assembly and binding to GSK3β and 14-3-3

Keratins are intermediate filament (IF) proteins expressed in various cell types, playing a crucial role in mechanical support and signaling pathways. Keratin 19 (K19) is predominantly found in gut epithelia and early embryonic ectoderm. K19 interacts with signaling proteins such as GSK3β and 14-3-3 to participate in cell signaling pathways. However, details on the binding sites of these proteins on K19, and specific molecular mechanisms governing these interactions, are lacking. Additionally, the N-terminal head domains of IFs including K19 are considered intrinsically disordered regions (IDRs), and their atomic-resolution structures are unavailable. Using NMR spectroscopy, we obtained the first solution structures of K19 head domain peptides. Structural models involving phosphomimetic peptides suggest a phosphorylation-dependent conformational change in the K19 head domain. The phosphomimetic S35E substitution alters the conformation of the head domain, affecting its association with keratin filaments and K19-binding partners. NMR spectral changes indicate that the S35E substitution affects the association of the K19 head domain to K8/K19 filaments and GSK3β, whereas no significant binding changes were observed with 14-3-3ζ and 14-3-3σ. Cell-based analyses further showed that the S35E mutation altered keratin filament organization and reduced the proximity between K19 and GSK3β in 293T cells, whereas no significant change was observed in the proximity between K19 and 14-3-3σ. Our findings provide insight into the structure-function relationship of the head domains in IF proteins with direct implications for the assembly of filaments and their role in signal modulation.