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

To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild-type human keratin-1/keratin-10 helix 1B heterotetramer at 3.0 angstrom resolution. It revealed biochemical determinants for the A(11) mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10-1B heterodimer dictated tetramer assembly: the N-terminal hydrophobic pocket (defined by L227(K1), Y230(K1), F231(K1), and F234(K1)), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314(K1) and L318(K1)). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full-length filament assembly. Individual knob residue mutant F314A(K1), but not L318A(K1), abolished 1B tetramer formation. The K1-1B knob/pocket mechanism is conserved across keratins and many non-keratin intermediate filaments. To demonstrate how pathogenic mutations cause skin disease by altering filament assembly, we additionally determined the 2.39 angstrom structure of K1/10-1B containing a S233L(K1) mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1(S233L)/K10-1B in solution without affecting secondary structure. The K1(S233L)/K10-1B octamer structure revealed S233L(K1) causes aberrant hydrophobic interactions between 1B tetramers.