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

Abstract

Summary 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 relevant substrates.

An atlas of Arabidopsis protein S-Acylation reveals its widespread role in plant cell organisation of and function

“…The acyl group is added by a family of protein acyl transferases (PATs) that are characterised by a DHHC motif within their catalytic domain. The structure of a human PAT, DHHC17, has recently been solved 4 , but despite this and the identification of a large number of S-acylation sites, there are no recognisable motifs around the modified cysteines of the substrate proteins. A recent study that monitored S-acylation directly using mass spectrometry (MS) suggested that cysteines were modified independently of any sequence motif around the site, in a stochastic process that depends upon the accessibility of any given cysteine to the action of a PAT 5 .…”

The Linker Domain of SNAP25 Acts as a Flexible Molecular Spacer to Ensure Efficient S-Acylation

“…The affinity of the zDABM of SNAP25 for the ANK domain of zDHHC17 was calculated to be ~ 11 M, although full-length SNAP25 had a higher affinity [19]. This interaction affinity is clearly sufficient to allow robust isolation of the protein complex by co-IP.…”

“…Our previous work identified a consensus recognition short linear motif (SLiM) in these and other substrates that mediate binding to the ANK domain of both zDHHC17 and zDHHC13 [18]. The 6-amino acid [VIAP][VIT]XXQP consensus motif makes key contacts with asparagine-100 and tryptophan-130 of zDHHC17 [19]. We named the consensus SLiM the "zDHHC Ankyrin repeat Binding Motif (zDABM)" [20].…”

“…We named the consensus SLiM the "zDHHC Ankyrin repeat Binding Motif (zDABM)" [20]. The affinity of the SNAP25 zDABM for the ANK domain of zDHHC17 is ~11 M, although full-length SNAP25 has a higher affinity (0.5 M) [19], likely owing to optimisation of binding/presentation of the zDABM to zDHHC17. The presence of other zDHHC17 binding sites in SNAP25 is unlikely as mutation of proline-117 in the zDABM blocks binding to the ANK domain, S-acylation and membrane targeting [18,21,22].…”

“…An interesting observation that came from this analysis is that not all of proteins that contain zDABMs are S-acylated by zDHHC17 [20], suggesting that other features of the binding partner must dictate whether or not it is an effective S-acylation substrate. Although the crystal structure of the ANK domain of zDHHC17 (with and without substrate peptide) has been reported [19], there is currently no structural information available for the full-length enzyme. Thus, it is unclear how the substrate-binding ANK domain and the catalytic DHHC domain of zDHHC17 are positioned relative to each other.…”