Extended Data Fig. 5: GTP-induced assembly of GBP1F micelles and membrane fragmentation.
From: Structural basis of antimicrobial membrane coat assembly by human GBP1
(a) Micelle formation of GBP1F (black arrows) was observed after addition of 1 mM GTP to a highly concentrated GBP1F solution (119 µM) in the absence of lipids (left panel). Upon addition of 2 mg/ml SUVs, GBP1F remodelled SUVs into spherical micelles (middle panel; arrows) and short filaments (white asterisks). A sample containing 2 mg/ml of SUVs without GBP1F is shown for comparison (right panel). (b) Confocal fluorescence imaging of GBP1F on GUVs. GBP1F -Q577C-AF647 shows weak binding to Texas Red-DHPE labelled GUVs 3 min after the addition of 1 mM GTP (white arrows). Scale bars correspond to 5 µm. (c) Dual trap bead-supported membrane transfer assay. Shown are mean fold-changes in inter-bead fluorescence measured in the dual trap membrane transfer assay for GBP1 in the presence/absence of different guanosine nucleotides and for the GBP1-R48A mutant deficient in GTPase activity. Error bars represent standard deviations and significance levels were determined using a one-sided non-parametric Mann-Whitney U test (*P < 0.05, **P < 0.01, ***P < 0.001. Individual P values (all relative to GBP1-GTP): GBP1-GDP P = 0.00058; GBP1-GDPAlF3 P = 0.0025; GBP1-R48A P = 0.00054; GBP1 P = 0.0026; GTP P = 0.0021). (d) SEC-MALS profiles for non-farnesylated GBP1-R48A (left panel) and farnesylated (right panel) GBP1F -R48A showing that deficiency in GTPase activity prevents dimerisation. (e) GTPase activity assay for the GTPase activity-deficient GBP1-R48A and GBP1F -R48A. GTPase activity for wild-type GBP1 is shown for comparison. Low luminescence signal corresponds to high GTPase activity. Data are presented as mean values +/− standard deviation (n = 3). Statistical significance was determined using two-sided Welch’s t-test with Bonferroni correction (*P = 0.009, **** P = 2.7*10−8, ns = not significant). Bovine serum albumin (BSA) was used as a negative control. (f) Representative negatively stained micrograph of GBP1F -R48A in the presence of BPLE-SUVs. No coated SUVs could be observed.
