Binding of the periplakin linker requires vimentin acidic residues D176 and E187

摘要

Electrostatic surface potential and ribbon representation for the I-TASSER derived periplakin linker domain model (C-score = 0.99). Electrostatic surface potential was calculated with DelPhi with the potential scale ranging from −7 (red) to +7 (blue) in units of / . The putative vimentin docking site is shown (black dashed circle). The ribbon model shows a PR2-like motif (green) and an Nt PR-like module (grey), and highlights the position of residues (stick format) mutated in periplakin transfected cells. Two dimensional H, N-resolved NMR spectra of the periplakin linker domain. Constructs encoding residues M1588–K1756 of human periplakin with a C-terminal HA tag were transfected into HeLa cells. Cells were stained with anti-HA and anti-vimentin antibodies. Periplakin is stained purple and vimentin is stained green in the merged images. The boxed areas are expanded on the far right-hand side. M merged image, HA periplakin staining, V vimentin staining. Bars, 10 µm. Manders’ overlap coefficient (MOC) was calculated for each image and is shown as a Tukey box plot with the median and 25th and 75th percentiles of each distribution. An unpaired test with Welch’s correction was performed on the data: Wild-type (WT) versus R1655E/R1656E,  = 0.002; WT versus K1687E/R1689E, not significant; WT versus R1713E/K1714E,  = 0.03; WT versus R1737E/K1741E,  = 0.03. At least five fields of view were analysed for each experiment. z-stacks were taken for each field and overlap coefficients calculated for each individual z-stack. An average overlap coefficient was then calculated for each experiment and each experiment was repeated two to three times.