Fig. 5: Interaction Between Pro-σK and the SpoIVFB Interdomain Linker.
From: Substrate engagement by the intramembrane metalloprotease SpoIVFB
A View of the interaction between the SpoIVFB interdomain linker (dark blue) and the alpha-helical cytosolic domain of Pro-σK (orange). The CBS domain of SpoIVFB is colored transparent green. B Anti-pentaHis western blot showing Pro-σK cleavage by SpoIVFB interdomain linker variants expressed in E. coli. Bands corresponding to SpoIVFB, Pro-σK, and processed σK are indicated to the right. Approximate locations of molecular weight markers are shown to the left. C Densitometry-based quantification of Pro-σK cleavage by SpoIVFB variants in the interdomain linker from (B). Bars represent the average cleavage ratio ([σK]/[Pro-σK + σK]) calculated from three (n = 3) independent experiments, with error bars representing standard error of the mean (SEM). Red circles indicate individual data points from the triplicate measurements. Significance from an unpaired two-sided t-test between each variant and WT is indicated with asterisks (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ****P ≤ 0.0001). P values for each variant are E44Q = 0.004, H203A = 0.7336, Y204A = 0.3242, H206A = 0.8544, R208A = 0.0520, F209A = 0.1382, E212A = 0.7979, R213A = 0.270, Y215A = 0.7856, Y204A/R208A = 0.0037. D Anti-pentaHis western blot showing in vivo photocrosslinking between SpoIVFB and Pro-σK. The position of amber stop codons in SpoIVFB or Pro-σK is indicated at the top. Crosslinked SpoIVFB species are indicated with an asterisk. Approximate molecular weight markers are shown to the left. The experiment was repeated once. E Close-up view of the interface between SpoIVFB and Pro-sK. Pairs of residues that showed strong disulfide crosslinking (F) are shown with red spheres and connecting dotted black lines. F Anti-FLAG western blots showing in vivo disulfide crosslinking between single-cysteine variants of SpoIVFB and Pro-sK. The position of cysteine residues in SpoIVFB and Pro-sK is indicated at the top, as is treatment with oxidant Cu2+(phenanthroline)3 (Cu) (1 mM) or reductant dithiothreitol (DTT) (100 mM) to promote or inhibit disulfide crosslinking, respectively. Crosslinked SpoIVFB species are indicated with an asterisk. The position of migration of protein molecular weight (MW) markers is shown. The experiment was repeated once.
