Department of Dermatology, Yale University, New Haven, CT, USA.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
EMBO J. 2019 Jun 3;38(11). doi: 10.15252/embj.2018100741. Epub 2019 Apr 29.
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 Å resolution. It revealed biochemical determinants for the A 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, Y230, F231, and F234), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314 and L318). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full-length filament assembly. Individual knob residue mutant F314A, but not L318A, 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 Å structure of K1/10-1B containing a S233L mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1/K10-1B in solution without affecting secondary structure. The K1/K10-1B octamer structure revealed S233L causes aberrant hydrophobic interactions between 1B tetramers.
为了在原子分辨率下描述角蛋白中间丝组装机制，我们确定了野生型人角蛋白-1/角蛋白-10 螺旋 1B 异四聚体的晶体结构，分辨率为 3.0Å。该结构揭示了角蛋白丝中 A 模式轴向排列的生化决定因素。K1/K10-1B 异二聚体疏水面上的四个区域决定了四聚体组装：N 端疏水性口袋（由 L227、Y230、F231 和 F234 定义）、K10 疏水性条纹、K1 相互作用残基和 C 端锚固旋钮（由 F314 和 L318 形成）。将两个旋钮残基突变为丙氨酸会破坏角蛋白 1B 四聚体和全长丝组装。单个旋钮残基突变 F314A，但不是 L318A，会导致 1B 四聚体形成。K1-1B 旋钮/口袋机制在角蛋白和许多非角蛋白中间丝中是保守的。为了证明致病突变如何通过改变丝组装引起皮肤疾病，我们还确定了含有与表皮松解性掌跖角化病相关的 S233L 突变的 K1/10-1B 的 2.39Å 结构。光散射和圆二色性测量表明，K1/K10-1B 在溶液中的聚集增强，而不影响二级结构。K1/K10-1B 八聚体结构表明 S233L 导致 1B 四聚体之间异常的疏水相互作用。
