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

Abstract

S-Acylation of the SNARE protein SNAP25 is mediated by a subset of Golgi zDHHC enzymes, in particular zDHHC17. The ankyrin repeat (ANK) domain of this enzyme interacts with a short linear motif known as the zDHHC ANK binding motif (zDABM) in SNAP25 (112-VVASQP-117), which is downstream of the S-acylated cysteine-rich domain (85-CGLCVCPC-92). In this study, we have investigated the importance of the flexible linker (amino acids 93-111; referred to as the "mini-linker" region) that separates the zDABM and S-acylated cysteines. Shortening the mini-linker had no effect of zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, whereas extended and rigid alanine-proline repeats perturbed this process. Indeed, 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 localisation as wild-type SNAP25, showing that the sequence of the mini-linker is not important in this context. By using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher affinity zDABM, and this mutant showed enhanced interaction with zDHHC17 in cells. Interestingly, this mutant was S-acylated with reduced efficiency, implying that a lower affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. Overall, the results of this study show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer to ensure efficient coupling of enzyme-substrate interaction and S-acylation.