Fig 2: BR187–385 binds to the tail-rod-2B end of KRT10. (a) BR187–385 bound to KRT10-FL immobilized on Ni-NTA magnetic agarose beads (+), and a small fraction remained associated after washing, while BR187–385 incubated with empty beads (−) was completely removed following the fourth wash step. Beads were analysed using SDS–PAGE before incubation with BR187–385 (0), after 10 min wash using magnetic PBS buffer (1), 1 min wash using magnetic washing buffer (2) and three times of a 10 min wash using magnetic washing buffer (3–5). The supernatant of BR187–385 after incubation with the beads (SN) confirms that equal amounts of protein were used for the assay. (b) A scheme of the tripartite structure of the intermediate filament protein keratin-10 (KRT10) shows the glycine loops localized at both end domains that are separated by a α-helical rod domain. The KRT10-FL, KRT10-HRD, KRT10-TRD and KRT10-ROD constructs comprise the stretches of residues 1–584, 1–179, 385–579 and 137–448, respectively. While BR187–385 bound to both KRT10-FL and KRT10-TRD with a common EC50-value of 345 nM (256–460 nM at 95% CI, R2 of 0.82), binding to the KRT10-rod domain (KRT10-ROD) was significantly reduced with an estimated EC50-value of 1.8 μM (1.4–2.3 μM at 95% CI, R2 of 0.74). Binding to the KRT10 head-region-domain was at a much lower level (KRT10-HRD). Values are given as mean with 95% CI. EC50 values were derived using a single four-parameter logistic nonlinear regression model. The mean normalized immobilization levels for KRT10-FL, KRT10-HRD, KRT10-TRD and KRT10-ROD as determined by HRP-coupled anti-His antibodies were 1.0 ± 0.2 (mean ± s.d.), 1.1 ± 0.4, 0.7 ± 0.2 and 1.1 ± 0.2, respectively.

Fig 3: BR187–385 comprises a paperclip-like region and a basic binding groove for interaction with KRT10. (a) The putative paperclip region provides an explanation for previous experimental observations [9]. The back- and front-loops of the clip, formed by residues 268–295 and 305–324, are coloured red and orange, respectively. Boxes highlight this specific region. Binding of KRT10 most probably requires conformational re-arrangements of the three loops LC1/C2, LD1/D2 and LD3/D4 (see electronic supplementary material, figure S4). (b) Hydrophobic patches on BR187–385 are displayed on a surface hydrophobicity distribution plot. Regions coloured red and blue are hydrophobic (positive values) and hydrophilic (negative values), respectively. Two hydrophobic patches are localized within the KRT10-binding region. While hydrophobic patch-1 is localized proximally to strand D4, hydrophobic patch-2 is localized underneath the tip of the front-loop of the clip. Hydrophobicity is plotted on a relative scale. (c) Analysis of the surface electrostatic potential of BR187–385 reveals a highly positively charged binding groove formed by the extended antiparallel β-sheet I that could accommodate the acidic helical rod domain of the KRT10/KRT1 heterodimer (see electronic supplementary material, figure S5). The electrostatic potential is plotted in with the Boltzmann's constant kb, the charge of an electron ec at a temperature of 298 K.

Fig 4: KRT10-TRD is bound in two contiguous paperclip-associated binding sites within BR187–385. (a) The location of each substituted residue is indicated. The centre of mass of each residue is displayed as a sphere and coloured according to its importance for binding to KRT10-TRD (red and blue for low and high binding intensities, respectively). The introduced substitutions did not significantly alter the overall secondary structure of the mutated BR187–385 proteins (see electronic supplementary material, figure S7). (b) Normalized intensity values were determined for binding of WT BR187–385 and each mutated variant to KRT10-TRD coated in wells of ELISA plates. Values are given as mean with standard deviation. WT and mutated BR187–385 variants were classified into groups a–d using the Tukey test at a significance level of p < 0.05.