Keratin 17 tail domain has a role in keratin 6/17 filament formation

Abstract

Keratins are the largest subgroup of intermediate filament (IF) proteins, which form 10-nm filaments with type I/II heterodimers in epithelial cells. The keratin 6 (K6) and keratin 17 (K17) pairs (K6/K17) are stress-induced keratins specifically expressed when wounds occur on the skin and play a key role in driving wound healing. To perform these functions, keratins require the assembly and bundling of filaments. It has been known that the rod domain plays a major role in forming this filament structure, and tail domains are not required for filament formation. For example, a tail deletion mutant of the K14, the basal layer-specific keratin, can form 10-nm filaments indistinguishable from wild-type keratins. In this study, however, we suggest that the K17 tail domain (K17T) plays a significant role in filament structure formation in the case of K6/K17. In this report, we used recombinant human proteins, including wildtype K6, wildtype K17, K17 rod domain (K17R), and tail-less K17 (K17ΔT), which were purified and reconstituted as described in previous works of literature. We conducted in vitro filament assembly studies on K6/K17, K6/K17R, and K6/K17ΔT pairs. To verify the abilities for filament structure formation among the groups, a high-speed sedimentation assay with Air-driven ultracentrifuge (Airfuge) was used. Using Transmission Electron Microscope (TEM), we directly observed the filament formation patterns of each group. Also, to elucidate the mechanism of how K17T influences K6/17 filament formation, we conducted a binding assay for K17T using a cross-linker, disuccinimidyl suberate (DSS). Furthermore, for structural analysis of K17T, NMR experiments with 13C- & 15N-labeled K17T were conducted. In the high-speed sedimentation assay using Airfuge, the K6/K17 group showed that most of the proteins were retrieved from the pellet. However, the K6/K17ΔT group displayed only about half of the proteins in the pellet. The K6/K17R group also revealed that most of the proteins were found in the supernatant. Using TEM, we observed that the K6/K17 group exhibited an elongated filament structure, while the K6/K17ΔT group displayed truncated filaments with the presence of spherical particles. In the case of the K6/K17R group, it did not exhibit any filamentous structures, only spherical particles. In the binding assay for K17T using the cross-linker DSS, K17T formed self-dimerization. In the structural analysis using NMR experiments, we discovered that K17T exhibited a high level of disorder. In the High-speed sedimentation assay, the majority of K6/K17 proteins were localized in the pellet, whereas the K6/K17ΔT group exhibited about half of the proteins in the pellet. This suggests that although the keratin 17 tail is not indispensable, it significantly influences keratin filament formation, as also confirmed by TEM imaging. TEM observations further confirmed that the K6/K17R pair failed to generate filaments and only formed particles, highlighting the insufficiency of the K17 rod alone for K6/K17 filament assembly. Additionally, with the presence of shorter filaments and spherical particles in the K6/K17ΔT group, we can infer the contributory roles of the K17T in both filament formation and stabilization. As the mechanism of how the K17T stabilizes the K6/K17 filament structures, we suggest interactions between the K17T, as confirmed by the binding assay with DSS. We also conducted a structural analysis for the K17T, and we have confirmed that K17T exhibits intrinsically disordered protein (IDP) characteristics.