Figure S5.
Effect of mutations in N-terminus or loop on ABHD17A conformation. (A) The N-terminus and nearby loop, which are responsible for the binding of ABHD17A to the membrane, are localized at the entrance of the hypothesized substrate binding pocket. (B) The probability distributions of the distance between the N-terminus and loop from AA simulations, in water versus bound to the membrane (upper left), indicate that the opening of the cavity displays conformational flexibility, which could influence substrate entry/exit. When the N-terminal cysteine residues are mutated to serine (lower left) or the hydrophobic loop residues are mutated to alanine (lower right), the N-terminus and loop regions are more dynamic, likely due to decreased membrane binding. When the hydrophobicity of the N-terminus is restored in the 5C>W/L mutant (upper left), the enzyme displays a smaller range of conformations, which may be optimal for substrate binding. Histograms were smoothed using the kernel density estimation method. (C) Individual mutation of hydrophobic loop residues to alanine has little effect on ABHD17A activity. GFP-NRas acylation was detected by 17-ODYA click-labeling. Levels of immunoprecipitated NRas (middle panel), or total ABHD17A (lower panel), were detected by western blot using anti GFP or FLAG antibody, respectively. (D) Quantification of NRas acylation in C. Acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, * = P ≤ 0.05. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (E) Individual mutation of the hydrophobic loop residues to alanine has a small effect on substrate binding in cABPP analysis. (F) Quantification of E depicted by percentage of IDFP inhibition. One-way ANOVA with Tukey’s multiple comparison test; n = 3, * = P ≤ 0.05, ** = P ≤ 0.01. Statistical analysis shows comparison to WT ABHD17A. Error bars indicate STDEV. Source data are available for this figure: SourceData FS5. Refer to the image caption for details.

Effect of mutations in N-terminus or loop on ABHD17A conformation. (A) The N-terminus and nearby loop, which are responsible for the binding of ABHD17A to the membrane, are localized at the entrance of the hypothesized substrate binding pocket. (B) The probability distributions of the distance between the N-terminus and loop from AA simulations, in water versus bound to the membrane (upper left), indicate that the opening of the cavity displays conformational flexibility, which could influence substrate entry/exit. When the N-terminal cysteine residues are mutated to serine (lower left) or the hydrophobic loop residues are mutated to alanine (lower right), the N-terminus and loop regions are more dynamic, likely due to decreased membrane binding. When the hydrophobicity of the N-terminus is restored in the 5C>W/L mutant (upper left), the enzyme displays a smaller range of conformations, which may be optimal for substrate binding. Histograms were smoothed using the kernel density estimation method. (C) Individual mutation of hydrophobic loop residues to alanine has little effect on ABHD17A activity. GFP-NRas acylation was detected by 17-ODYA click-labeling. Levels of immunoprecipitated NRas (middle panel), or total ABHD17A (lower panel), were detected by western blot using anti GFP or FLAG antibody, respectively. (D) Quantification of NRas acylation in C. Acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, * = P ≤ 0.05. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (E) Individual mutation of the hydrophobic loop residues to alanine has a small effect on substrate binding in cABPP analysis. (F) Quantification of E depicted by percentage of IDFP inhibition. One-way ANOVA with Tukey’s multiple comparison test; n = 3, * = P ≤ 0.05, ** = P ≤ 0.01. Statistical analysis shows comparison to WT ABHD17A. Error bars indicate STDEV. Source data are available for this figure: SourceData FS5.

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