The X-Ray Crystal Structure of the Keratin 1-Keratin 10 Helix 2B Heterodimer Reveals Molecular Surface Properties and Biochemical Insights into Human Skin Disease

Abstract

Keratins 1 (K1) and 10 (K10) are the primary keratins expressed in differentiated epidermis. Mutations in K1/K10 are associated with human skin diseases. We determined the crystal structure of the complex between the distal (2B) helices of K1 and K10 to better understand how human keratin structure correlates with function. The 3.3Å resolution structure confirms many features inferred by previous biochemical analyses but adds new, unexpected insights. It demonstrates a parallel, coiled-coil heterodimer with a predominantly hydrophobic intermolecular interface; this heterodimer formed a higher order complex with a second K1-K10-2B heterodimer via a Cys401K10 disulfide link, although the bond angle is unanticipated. Molecular surface analysis of K1-K10-2B identified several pockets, one adjacent to the disulfide linkage and conserved in K5-K14. The solvent accessible surface area of the K1-K10 structure is 20-25% hydrophobic. The 2B region contains mixed acidic and basic patches proximally (N-terminal) whereas it is largely acidic distally (C-terminal). Mapping of conserved and non-conserved residues between K1-K10 and K5-K14 onto the structure demonstrated the majority of unique residues align along the outer helical ridge. Finally, the structure permitted a fresh analysis of the deleterious effects caused by K1/K10 missense mutations found in patients with phenotypic skin disease.

Human keratin 1/10‐1B tetramer structures reveal a knob‐pocket mechanism in intermediate filament assembly

“…The electrostatic surface potential of the K1/K10‐1B heterodimer is similar to that observed in the K1/K10‐2B heterodimer (Bunick & Milstone, ): There is polarization of charge with the distal three‐fourths of the complex being acidic, whereas the proximal one‐fourth is more basic (Fig C). The basic patch at the N‐terminus of K1/K10‐1B contains residues from both K1 (R239, R240, R241) and K10 (K198, K201, K207; Fig D); this is in contrast to the 2B heterodimer, where a linear N‐terminal basic patch was solely formed by nine K1 residues (Bunick & Milstone, ).…”

“…The molecular surfaces of IFs contain features critical to their assembly and function. Perhaps the most important finding from the previous K1/K10‐2B heterodimer structure was that only a small number of residue differences are needed to significantly alter the shape and chemistry of the keratin surface because most of the unique residues concentrate along the outer helical ridges of the coiled‐coil (Bunick & Milstone, ). This holds true for K1/K10‐1B as well (Figs EV3A and B, and EV4A–D).…”

Keratin 14-dependent disulfides regulate epidermal homeostasis and barrier function via 14-3-3σ and YAP1

“…Three lines of evidence support this contention. First, Bunick and Milstone (Bunick and Milstone 2017) succeeded in crystallizing the interacting 2B segments of K1-K10, the keratin pairing characteristic of differentiating keratinocytes in epidermis (Fuchs 1995). The K1(2B)-K10(2B) structure (PDB 4ZRY) show an identical overall fold to our the original K5(2B)-K14(2B) structure , including the presence of a trans-dimer, homotypic disulfide bond mediated by the stutter cysteine (C401) in K10 (Bunick and Milstone 2017).…”

Desmosomes and Intermediate Filaments: Their Consequences for Tissue Mechanics

“…With the exception of KRT8, KRT18, and KRT19, all other human and mouse keratins contain variable numbers of cysteine residues. Following the observation that a subset of keratins becomes disulfide-crosslinked on terminal differentiation in vivo, homotypic disulfide bonds involving three Cys residues in KRT14 and one in K10 were identified in cultured keratinocytes and mouse epidermis, in addition to interkeratin disulfide-bonded KRT5/14 species Feng and Coulombe 2015;Bunick and Milstone, 2016). Available data point to multiple roles of disulfidebonding on keratin assembly, dynamics, and network organization.…”