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 3.3 Å resolution structure confirms many features inferred by previous biochemical analyses, but adds 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 Cys401^K10 disulfide link, although the bond angle is unanticipated. The 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 nonconserved 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.

Fig. 1. Disulfide bonding in the keratin 1-keratin 10-2B crystal structure

(a) K1 and K10 domain organization, which is conserved among KIFs, features four helical segments (1A, 1B, 2A, 2B) flanked by variable head and tail regions. (b) The K1-K10-2B heterodimer formed a trans-dimer, homotypic disulfide bond via Cys401^K10. The disulfide bond occurred between K10 molecules in different, adjacent asymmetric units of the crystal lattice. No other cysteine in K1-K10-2B was observed to form disulfide bonds. (c) Rotated global view, compared to panel A, of the disulfide bonded K1-K10 complex; a zoomed in view (boxed) of the residues surrounding the disulfide bond demonstrate Gly398^K10 and Ser405^K10 are structurally adjacent to Cys401^K10. (d) The carbonyl oxygen of Cys401^K10 is 2.7 Å from Ser405^K10 hydroxyl oxygen and 3.0 Å from Ser405^K10 amide nitrogen. (e) Multiple sequence alignment of the K10-2B stutter region (denoted ‘xxxx’) from various species and several type I keratins from Homo sapiens, demonstrating sequence differences in key positions related to disulfide bond formation. (f) Structural consequences of Leu437Pro^K1 mutation include loss of hydrophobic interactions with Tyr400^K10, Gln403^K10, and Leu404^K10 and loss of hydrogen bonds with Gln403^K10 adjacent to Cys401^K10; proline may also cause distal helix kinking. (g) Molecular surface representation of the disulfide-linked complex between two K1-K10-2B heterodimers highlighting symmetric concavities (yellow) adjacent to the disulfide bond (*). In vivo, K10 molecules located on the outside of the 10-nm filament will have Cys401^K10 exposed and able to form disulfide linkages; it is plausible disulfide-linked K1-K10 influences nucleus behavior and shape in differentiated keratinocytes based on K5-K14 studies (Feng and Coulombe, 2015, Lee et al., 2012).

Fig. 2. Electrostatic surface properties of K1-K10-2B

(a) K1-K10-2B heterodimer depicted in two orientations, related by 180°, demonstrating the 2B helix has polarization of charge: a mixed acidic and basic character at the N-terminus and predominantly acidic character at the C-terminus. (b) The N-terminal surface of K1-K10-2B is more acidic than the basal keratin complex K5-K14-2B because it contains a glutamate (Glu397^K10) rather than lysine (Lys363^K14) at homologous positions (circle). The K1-K10 and K5-K14 molecules are structurally aligned in the panel; there are 27 additional N-terminal residues observed in the K1-K10 structure. (c) Acidic patch formed along the N-terminal surface of K10-2B. (d) Acidic pocket formed between Glu397^K1 and Glu400^K1.

Fig. 3. Molecular surface properties of K1-K10-2B

(a) K1-K10-2B heterodimer depicted in two orientations, related by 180°, demonstrating it contains several solvent accessible and surface exposed hydrophobic residues. Non-hydrophobic residues are colored blue. Hydrophobic residues are colored light (less) to dark (more) orange, based on degree of hydrophobicity. (b) Nature of the solvent accessible surface area of the K1-K10-2B heterodimer. (c, d) Concavities/pockets in the molecular surface of K1-K10-2B (gold) and K5-K14-2B (green). Both Cys367^K14 and Cys401^K10 (cysteines colored yellow) have a pocket adjacent to it; the chemical nature of the pockets is mostly conserved. One pocket wall is formed by Lys363 in K14 (basic) but is formed by Glu397 in K10 (acidic). (e) Residues in K1 (green) and K10 (magenta) that are unique (non-conserved) compared to K5 and K14, respectively, are mapped onto the K1-K10-2B structure, demonstrating alignment mostly along outer helical ridges. Conserved residues align mostly along the helical interface (right upper box), with some regions appearing groove-like (*). Some pockets, such as the ‘Central’ pocket in panel C, have conserved residues (Ile421^K10 and Thr425^K10) lining the floor.

Fig. 4. Structural consequences of K1-K10-2B mutations

(a) Leu452Pro^K10 mutation associated with epidermolytic ichthyosis eliminates a surface-exposed leucine and generates proline ring clashes leading to helix kinking. (b) Ile479Phe^K1, associated with epidermolytic ichthyosis, generates steric clashes from the phenylalanine aromatic ring; Ile479Thr^K1, associated with epidermolytic ichthyosis and epidermolytic palmoplantar keratosis, fails to provide the same stabilizing hydrophobic interactions or hydrogen bonds that wild-type isoleucine provides. (c) Leu485Pro^K1 mutation associated with cyclic ichthyosis with epidermolytic hyperkeratosis eliminates hydrophobic interactions (with L453^K10) and generates clashes between proline and the carbonyl oxygen of Tyr482^K1 that drives kinking of the K1-2B helix. (d) All human keratin mutations (for K1, K5, K6a, K6b, K6c, K10, K14, K16, K17) documented in the Human Intermediate Filament Database were analyzed for the type of chemical change occurring and results for K1/K10 are summarized. The most common type of change was a hydrophobic residue to a hydrophobic residue (26%), and second most common was charged residue to a polar residue (14%). Definitions of residues were: acidic (D, E), basic (R, K), polar (Q, N, H, S, T, Y, C, G) and hydrophobic (A, I, L, F, V, P, M, W). Data for K5-K14 and pachyonychia congenita keratins are summarized in Figure S14.