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

Abstract

DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However how DHHC enzymes engage with their substrates is poorly understood. There is currently no high-resolution structural information for the interaction between any DHHC enzyme and protein substrates. Among the DHHC family, DHHC17 and DHHC13 have special features: they both have six transmembrane helices and a large cytoplasmic domain with seven ankyrin repeats (ANK). The ANK domain is known to harbor the binding site for several substrates. DHHC17, also known as Huntingtin Interacting Protein 14, is highly expressed in neurons and acts on substrates such as Snap25b and Huntingtin. The importance of DHHC17 and DHHC13 is underscored by the fact that loss of both lead to embryonic lethality in mice. In this study we investigate the structural and thermodynamic bases of the interaction between the ANK domain of DHHC17 (ANK17) with Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment of Snap25b containing the recognition motif and use a combination of binding studies in vitro (with purified proteins) and in vivo to validate the structural model. Our structure reveals that the N-terminal part of the peptide substrates makes key contacts with DHHC17 and through mutagenesis analysis we discovered single point mutations in DHHC17 that completely prevent binding to Snap25. Finally, we extend our study to the interaction between ANK17 and Huntingtin, one of the most prominent substrates of DHHC17. Overall, our research represent a first step for the elucidation of the mechanism of action of DHHC17 and it will be valuable toward the development of regulators of DHHC17 function.