Figure 4.
Hydrophobic loop residues are important for activity. (A) Schematic of the alanine scan used to determine the regions of the loop required for ABHD17A activity. (B) Alanine loop mutants show flanking regions of the loop are important for ABHD17A activity on NRas. Activity of ABHD17A was measured by acylation of GFP-NRas as detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (middle panel), or total ABHD17A (lower panel), were detected by western blot using anti-GFP or FLAG antibody, respectively. (C) Quantification of GFP-NRas acylation in B. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (D) AF2 predicted structure of ABHD17A showing the loop adjacent to the active site in blue. Inset shows a close-up of the loop and the orientation of the side chains of the three hydrophobic loop residues F222, Y229, and F231. (E) Three hydrophobic loop residues (F222, Y229, and F231) are needed for ABHD17A activity on GFP-NRas. Acylation of GFP-NRas was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (middle panel), or total ABHD17A (lower panel), were detected by western blot using anti-GFP or FLAG antibody, respectively. (F) Quantification of GFP-NRas acylation in E. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (G) Insertion depth analysis shows that mutating hydrophobic loop residues 222, 229, 231 to alanine in AA simulations of acylated ABHD17A impairs membrane insertion of the conserved loop. Source data are available for this figure: SourceData F4. Refer to the image caption for details.

Hydrophobic loop residues are important for activity. (A) Schematic of the alanine scan used to determine the regions of the loop required for ABHD17A activity. (B) Alanine loop mutants show flanking regions of the loop are important for ABHD17A activity on NRas. Activity of ABHD17A was measured by acylation of GFP-NRas as detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (middle panel), or total ABHD17A (lower panel), were detected by western blot using anti-GFP or FLAG antibody, respectively. (C) Quantification of GFP-NRas acylation in B. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (D) AF2 predicted structure of ABHD17A showing the loop adjacent to the active site in blue. Inset shows a close-up of the loop and the orientation of the side chains of the three hydrophobic loop residues F222, Y229, and F231. (E) Three hydrophobic loop residues (F222, Y229, and F231) are needed for ABHD17A activity on GFP-NRas. Acylation of GFP-NRas was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (middle panel), or total ABHD17A (lower panel), were detected by western blot using anti-GFP or FLAG antibody, respectively. (F) Quantification of GFP-NRas acylation in E. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (G) Insertion depth analysis shows that mutating hydrophobic loop residues 222, 229, 231 to alanine in AA simulations of acylated ABHD17A impairs membrane insertion of the conserved loop. Source data are available for this figure: SourceData F4.

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