Crystal Structure of Keratin 1/10(C401A) 2B Heterodimer Demonstrates a Proclivity for the C-Terminus of Helix 2B to Form Higher Order Molecular Contacts.

Publication Type:

Journal Article

Source:

Yale J Biol Med, Volume 93, Issue 1, p.3-17 (2020)

Keywords:

Amino Acid Sequence, Cytoskeletal Proteins, Cytoskeleton, Disulfides, Genetic Linkage, Humans, Intermediate Filaments, Keratin-1, Keratinocytes, Mutation, Peptide Fragments, Protein Conformation

Abstract:

<p>We previously determined the crystal structure of the wild-type keratin 1/10 helix 2B heterodimer at 3.3 Å resolution. We proposed that the resolution of the diffraction data was limited due to the crystal packing effect from keratin 10 (K10) residue Cys401. Cys401 formed a disulfide-linkage with Cys401 from another K1/10 heterodimer, creating an &quot;X-shaped&quot; structure and a loose crystal packing arrangement. We hypothesized that mutation of Cys401 to alanine would eliminate the disulfide-linkage and improve crystal packing thereby increasing resolution of diffraction and enabling a more accurate side chain electron density map. Indeed, when a K10 Cys401Ala 2B mutant was paired with its native keratin 1 (K1) 2B heterodimer partner its x-ray crystal structure was determined at 2.07 Å resolution; the structure does not contain a disulfide linkage. Superposition of the K1/K10(Cys401Ala) 2B structure onto the wild-type K1/10 2B heterodimer structure had a root-mean-square-deviation of 1.88 Å; the variability in the atomic positions reflects the dynamic motion expected in this filamentous coiled-coil complex. The electrostatic, hydrophobic, and contour features of the molecular surface are similar to the lower resolution wild-type structure. We postulated that elimination of the disulfide linkage in the K1/K10(Cys401Ala) 2B structure could allow for the 2B heterodimers to bind/pack in the A tetramer configuration associated with mature keratin intermediate filament assembly. Analysis of the crystal packing revealed a half-staggered anti-parallel tetrameric complex of 2B heterodimers; however, their register is not consistent with models of the A mode of tetrameric alignment or prior biochemical cross-linking studies.</p>

PDB: 
6UUI
Detector: 
PILATUS
Beamline: 
24-ID-C