The linker domain of the SNARE protein SNAP25 acts as a flexible molecular spacer that ensures efficient S-acylation

Christine Salaun, Jennifer Greaves, Nicholas C O Tomkinson, Luke H Chamberlain

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Abstract

S-Acylation of the SNARE protein SNAP25 (synaptosomeassociated protein of 25 kDa) is mediated by a subset of Golgi zinc finger DHHC-type palmitoyltransferase (zDHHC) enzymes, particularly zDHHC17. The ankyrin repeat domain of zDHHC17 interacts with a short linear motif known as the zDHHC ankyrin repeat- binding motif (zDABM) in SNAP25 ( 112VVASQP 117), which is downstream of its S-acylated, cysteine-rich domain ( 85CGLCVCPC 92). Here, we investigated the importance of a flexible linker region (amino acids 93-111, referred to hereafter as the “mini-linker” region) that separates the zDABM and S-acylated cysteines in SNAP25. Shortening the mini-linker did not affect the SNAP25-zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, but extended and rigid alanine-proline repeats perturbed it. A SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localization as WT SNAP25, indicating that the amino acid composition of the mini-linker is not important for SNAP25 localization. Using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher-affinity zDABM. This mutant interacted with zDHHC17 more strongly but was S-acylated with reduced efficiency in HEK293T cells, implying that a lower-affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. These results show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer that ensures efficient coupling of the SNAP25-zDHHC17 interaction and S-acylation of SNAP25.

Original languageEnglish
Pages (from-to)7501-7515
Number of pages15
JournalJournal of Biological Chemistry
Volume295
Issue number21
Early online date21 Apr 2020
DOIs
Publication statusPublished - 22 May 2020

Bibliographical note

Final version open access under the terms of the Creative
Commons CC-BY license

Keywords

  • S-acylation
  • zDHHC17
  • zDHHC enzyme
  • ankyrin repeat domain
  • mPEG-click
  • protein palmitoylation
  • protein domain
  • post-translational modification (PTM)
  • protein acylation
  • protein chemical modification

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