Extended Data Fig. 7: A C-terminal motif in FacZ is required for function and interaction with GpsB.
a, b) Gel filtration assays testing FacZ-GpsB interactions. Left: Binding assays performed with SUMO-3x-FacZ (127–146) containing the native binding motif (NRHYRR); Right: FacZ Binding assays performed with SUMO-3x-FacZ (127–146) containing an altered motif (NDHYDD). A) Gel filtration profiles of GpsB (1–75) alone (green), SUMO-3x-FacZ (127–146) alone (red), or a mixture of the two proteins (binding, blue). The sum of GpsB (1–75) alone (green) and SUMO-3x-FacZ (127-146) is shown in gray for display purposes. Elution profiles are representative of 6 runs. B) Representative SDS-PAGE gels of fractions from the size-exclusion column from runs with FacZ alone (top), GpsB alone (middle), and a mixture of both proteins (bottom). c) Diagram of FacZ C-terminus with proposed GpsB-interaction motif (yellow box) and the location of four arginine residues (bolded red R). d) Various R → D FacZ alleles were expressed from Plac on a pLOW vector in a ∆facZ background of strain RN4220, and then plated with or without PC19 as indicated. Disruption of R1 [aTB568], R2 [aTB570], or R3 [aTB572] alone had little impact on growth on PC19, while inactivation of all three together (3R → 3D) [aTB565] did not enable growth on PC19. Disruption of R4 [aTB574], outside of the proposed binding motif, had no impact on growth on PC19. e) Western blot of native FacZ [aTB347], FacZ-6x-His [aTB349], and FacZDDD (3R → 3D) [aTB651] expressed from a pLOW vector. f) Full blots of FacZ-His and GpsB-FLAG pulldowns from two representative replicates (see Fig. 6e, f).
