Structural Basis for Substrate Recognition by the Ankyrin Repeat Domain of Human DHHC17 Palmitoyltransferase

Abstract

DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are up to 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However, how DHHC enzymes engage with their substrates is still poorly understood. There is currently no structural information about the interaction between any DHHC enzyme and protein substrates. In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of human DHHC17 (ANK17) and Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment of Snap25b. Through structure-guided mutagenesis, we discovered key residues in DHHC17 that are critically important for interaction with Snap25b. We further extended our finding by showing that the same residues are also crucial for the interaction of DHHC17 with Huntingtin, one of its most physiologically relevant substrates.

Keywords: DHHC; DHHC17; Huntingtin; Snap25b; X-ray crystallography; palmitoylation; palmitoyltransferase; posttranslational modification; protein lipidation; protein-protein interaction.

Figure 1. Initial characterization of ANK17 binding to Snap25b

(A) Schematic topology of DHHC17 palmitoyl-transferase. (B) Two-step reaction scheme for a typical DHHC enzyme. (C) List of constructs used for crystallization: ANK17 (corresponding to N-terminal domain of Human DHHC17 - residues 51–288) and full-length Snap25b-WT were produced by recombinant expression in E.coli. Peptide corresponding to amino acids 111 to 120 in Snap25 was produced by solid phase peptide synthesis (D) Representative ITC thermograms and non-linear regression analyses for the interaction between ANK17 and Snap25b (left panel), Snap25b-C4A (central panel) or S25b_111–120 (right panel).

Figure 2. Crystal structure of the complex between ANK17 and Snap25_111–120

A–B) Cartoon and stick representation of the ANK17 and Snap25b peptide, respectively. C–D) Surface representation of the complex. The peptide (green surface) binds to the concave region of ANK17 between AR1–AR3. Some of the interacting residues in ANK17 are shown in orange.

Figure 3. ITC titration of ANK17 in Snap25b

A) ANK17-WT or ANK17-mutants titrated into Snap25b. ANK17-N100A and ANK17-W130A showed only heat of dilution. (B) ANK17-WT titrated into Snap25b mutants. Snap25b-P117A showed only marginal heat of binding and we could not obtain any fitting of the integrated heats. (C) Position of mutated residues in ANK17 and Snap25b in the structure of the complex. (D) Histogram representing binding affinity (K_d) determined by ITC titrations (n.d.: not determined).

Figure 4. Pull-down assay to determine affinity of DHHC17 to Snap25b and effect of single-point mutations

(A) Schematic depiction of the assay (B) 10xHis-GFP-DHHC17 and GFP-Snap25b-WT or mutants after 24 hours co-transfection in HEK-293T cells. Load: expression levels of each protein after lysis but prior to affinity purification. Co²⁺ affinity pull down: protein eluted from affinity resin after incubation for 16 hours and extensive washing. (C) Quantitation of the experiment in (B) after gel-densitometry analysis. The intensity of the band corresponding to Snap25b was divided the intensity of the corresponding DHHC17 band. Histograms represent average ± standard deviation (SD). One-way ANOVA with Dunnett’s multiple comparison test was used to compare mutants with WT (n.s., p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001). (D) and (E) Same as in (B) and (C), except that mutants of DHHC17 were used to pull-down Snap25b-WT. (F) Alignment of the first three ankyrin repeats of DHHC17 in different organisms and Human DHHC13. The conserved N100 and W130 residues are highlighted in blue. Sequence alignment was preformed using Clustal-omega (Sievers et al., 2011).

Figure 5. S-acylation of EGFP-Snap25b by DHHC17 and effect of DHHC17 mutations

HEK-293T cells were transfected with EGFP-Snap25b, EGFP-Snap25b-C4A (cysteine-less) or empty vector and GFP-DHHC17 or GFP-DHHC17 mutants. Cells were incubated with 17-ODYA fatty acid alkyne for 4 h at 37°C. S-acylation with fatty acid alkyne was detected by coupling the alkyne fatty acid to Rhodamine-azide dye via click chemistry reaction. Isolated proteins were resolved by SDS/PAGE and transferred to PVDF membranes. (A) Chemical structures of 17-ODYA (17-Octadecynoic Acid) and palmitic acid. (B) Representative image of a typical experiment is shown. Click chemistry signal (Top), anti-GFP immunoblot (Middle), and anti-Snap25 immunoblot (Bottom). (C) van der Waals contacts between W130 and P117. Contact dots were obtained using the method of Richardson and co. (Word et al., 1999). (D) Quantification of Snap25b S-acylation. The intensity of the Snap25b band in the immunoblot was divided by the intensity of the Snap25b band in the rhodamine detection gel. Histograms represent average ± standard deviation (n=3). One-way ANOVA with Dunnett’s multiple comparison test was used to compare mutants with WT (n.s., p ≥ 0.05, *p < 0.05).

Figure 6. ITC titrations for the binding of ANK17 to Huntingtin N-terminal region

(A) Constructs used in the titrations. ANK17 titrated into (B) MBP-Htt_1–548, (C) MBP-Htt_1–548-Q500A-P501A, (D) MBP-Htt_1–492. (E) ANK17-W130 titrated into MBP-Htt_1–548 (F) Peptide corresponding to amino acids 493–504 titrated into ANK17.