Fig. 4: Binding of the Pro-sequence of Pro-σ^K to SpoIVFB.

A Two views of a single monomer of the WT SpoIVFB:Pro-σ^K complex. SpoIVFB is colored blue, Pro-σ^K is colored orange, LMNG detergent is colored magenta with the corresponding cryo-EM map shown in magenta mesh. The gray box approximates the lipid membrane. The pro-sequence of Pro-σ^K engages the reentrant loop of SpoIVFB through b-sheet augmentation. The LMNG detergent molecule is positioned between TM1 and TM6, directly over the reentrant loop. B Close-up view of the Pro-σ^K pro-sequence engaging SpoIVFB through b-sheet augmentation. Individual residues of the pro-sequence are indicated. The three zinc-coordinating residues and catalytic E44 of SpoIVFB are highlighted in darker blue. The reentrant loop of SpoIVFB and TM1 are deleted from the image on the right to reveal the enzyme active site. C Schematic showing the amino acid sequence at the N-terminus of Pro-σ^K. The site of SpoIVFB-mediated cleavage is highlighted in red. D Anti-pentaHis western blot showing Pro-σ^K cleavage by SpoIVFB variants expressed in E. coli. The bands corresponding to SpoIVFB, Pro-σ^K, and processed σ^K are indicated to the left. Approximate locations of molecular weight markers are shown to the right. E Densitometry-based quantification of Pro-σ^K cleavage for the variants shown in (D). Bars represent the average cleavage ratio ([σ^K]/[Pro-σ^K + σ^K]) 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 = 0.001, L16W = 0.0119, V17W = 0.1737, F18W = 0.1565, L19W = 0.0051, V20W = 0.0254, S21W = 0.0001, Y22W = 0.0001, V23W = 0.0001, K24W = 0.0041.