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

Abstract

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 GSK3{beta} 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 GSK3{beta}, whereas no significant binding changes were observed with 14-3-3{zeta} and 14-3-3{sigma}. Cell-based analyses further showed that the S35E mutation altered keratin filament organization and reduced the proximity between K19 and GSK3{beta} in 293T cells, whereas no significant change was observed in the proximity between K19 and 14-3-3{sigma}. 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.