Fig. 4: Allosteric activation of DrrA by active Rab1.
From: Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1
a Construction of the DrrA apo-form. DrrA8−218 (PDB: 3NKU, orange) and DrrA193−352 (PDB: 3LOI, blue) are superimposed onto GS-ATase (PDB: 3K7D, light green background). Orange spheres represent the catalytic Asp residues of DrrA8−218. b Global structural changes in DrrA by binding to active Rab1. DrrA8−218 (PDB: 3NKU, orange) and DrrA16−352 (grey) are shown. c Structural changes in the active centre of DrrA induced by Rab1 binding to the non-conventional site. Purple spheres represent the catalytic centre of DrrA16−352 in the DrrA:Rab8a complex. Orange spheres represent the catalytic centre of DrrA8−218 (PDB: 3NKU, orange). d Sigmoidal dependence of AMPylation on the active Rab1 concentration. The red curve represents the Hill fit with a cooperativity parameter of n = 1.7 ± 0.16; the black curve is the Michaelis–Menten fit. Data are means ± SEM from three independent experiments. e Full- length DrrA16-647-mediated AMPylation. The red curve represents the Hill fit with a cooperativity parameter of n = 1.6 ± 0.20; the black curve is the Michaelis–Menten fit. Data are means ± SEM from three independent experiments. f Cytotoxicity analysis of DrrA single alanine mutants in H1299 cells. Cell viability values (determined by MTS assay) of DrrA-expressing cells (eGFP-positive) were determined in relation to the eGFP vector control. WT: DrrA8-533; R70A: DrrA8-533_R70A; Q71A: DrrA8-533_Q71A; K74A: DrrA8-533_K74A. Data are means ± SEM from three independent experiments. One-way analysis of variance (ANOVA) was applied; ns, p > 0.05; *0.01 < p < 0.05; ****p < 0.0001. Comparing to the wt, the p values from R70A, Q71A, K74A are 0.9172, 0.6747, and 0.1594 respectively. The p value between R70A and Q71A is 0.2671, the one between Q71A and K74A is 0.7575, and the one between R70A and K74A is 0.0454. g Cytotoxicity analysis of DrrA mutants in H1299 cells. Cell viability values (determined by MTS assay) of DrrA-expressing cells (eGFP-positive) were determined in relation to the eGFP vector control. TA: DrrA8-533_R70A_Q71A_K74A; DD: DrrA8-533_D110_D112A. Data are means ± SEM from three independent experiments. One-way analysis of variance (ANOVA) was applied; ns, p > 0.05; ***p < 0.001; ****p < 0.0001. Comparing the eGFP, the p values from wt, TA, and DD are 0.0005, 0.0002, and 0.9095, respectively. The p value between wt and DD is 0.0010. The p value between TA and DD is 0.0001. h Proximity-triggered crosslinking between BrC6K-bearing Rab1b and eGFP-DrrA8-533 mutants in living HEK293T cells as observed by α-GFP WB. Crosslinking is specific for DrrA-D82C and Rab1b-R69BrC6K for DD-DrrA variants; the corresponding triple alanine DrrA-variant (TA-DrrA) is deficient in crosslinking. Source data are provided as a Source Data file.
