Figure 5.
The N-terminus and loop affect binding pocket conformation. (A) Pocket analysis identifies one major cavity inside ABHD17A, indicated by the blue solid surface. Its localization close to the membrane interface suggests that the cavity is a potential substrate binding pocket for ABHD17A. (B–D) An ABHD17A surface model of the AF2 predicted structure reveals a prominent cleft. Palmitic acid is docked into this cleft by SwissDock (Grosdidier et al., 2011a). The active site serine is shown in yellow. (C) Close up of the top-scoring SwissDock model showing palmitic acid interacting with ABHD17A. (D) View of the membrane-contacting surface shows the N-terminus and loop are predicted to frame the entrance of the channel. (E) Schematic of the cABPP assay used to determine substrate binding of ABHD17A mutants. Cell lysates expressing ABHD17A-FLAG constructs are incubated in the presence or absence of 12-carbon fluorophosphonate inhibitor IDFP in DMSO. Subsequent incubation with the fluorescent activity probe TAMRA-FP covalently labels the catalytic serine of ABHD17A molecules that have not bound the inhibitor. IDFP binding causes a reduction of in-gel fluorescence (left side). Mutations that alter the conformation of the substrate-binding pocket reduce IDFP binding, thus no change in fluorescence is seen (right side). (F) The Src myristoylation motif paired with hydrophobic N-terminal residues partially restored the binding pocket conformation of non-acylated ABHD17A mutants. TAMRA-FP binding was visualized with in-gel fluorescence (upper panel) and total protein was detected by western blot using an anti-FLAG antibody (lower panel). (G) Quantification of F depicted by percentage of IDFP inhibition. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Statistical analysis shows the comparison to WT ABHD17A. Error bars indicate STDEV. (H) Mutation of three hydrophobic loop residues (F222, Y229, and F231) significantly decreased IDFP binding by cABPP analysis. (I) Quantification of H depicted by percentage of IDFP inhibition. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ** = P ≤ 0.01, *** = P ≤ 0.001. Statistical analysis shows comparison to WT ABHD17A. Error bars indicate STDEV. Source data are available for this figure: SourceData F5. Refer to the image caption for details.

The N-terminus and loop affect binding pocket conformation. (A) Pocket analysis identifies one major cavity inside ABHD17A, indicated by the blue solid surface. Its localization close to the membrane interface suggests that the cavity is a potential substrate binding pocket for ABHD17A. (B–D) An ABHD17A surface model of the AF2 predicted structure reveals a prominent cleft. Palmitic acid is docked into this cleft by SwissDock (Grosdidier et al., 2011a). The active site serine is shown in yellow. (C) Close up of the top-scoring SwissDock model showing palmitic acid interacting with ABHD17A. (D) View of the membrane-contacting surface shows the N-terminus and loop are predicted to frame the entrance of the channel. (E) Schematic of the cABPP assay used to determine substrate binding of ABHD17A mutants. Cell lysates expressing ABHD17A-FLAG constructs are incubated in the presence or absence of 12-carbon fluorophosphonate inhibitor IDFP in DMSO. Subsequent incubation with the fluorescent activity probe TAMRA-FP covalently labels the catalytic serine of ABHD17A molecules that have not bound the inhibitor. IDFP binding causes a reduction of in-gel fluorescence (left side). Mutations that alter the conformation of the substrate-binding pocket reduce IDFP binding, thus no change in fluorescence is seen (right side). (F) The Src myristoylation motif paired with hydrophobic N-terminal residues partially restored the binding pocket conformation of non-acylated ABHD17A mutants. TAMRA-FP binding was visualized with in-gel fluorescence (upper panel) and total protein was detected by western blot using an anti-FLAG antibody (lower panel). (G) Quantification of F depicted by percentage of IDFP inhibition. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Statistical analysis shows the comparison to WT ABHD17A. Error bars indicate STDEV. (H) Mutation of three hydrophobic loop residues (F222, Y229, and F231) significantly decreased IDFP binding by cABPP analysis. (I) Quantification of H depicted by percentage of IDFP inhibition. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ** = P ≤ 0.01, *** = P ≤ 0.001. Statistical analysis shows comparison to WT ABHD17A. Error bars indicate STDEV. Source data are available for this figure: SourceData F5.

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