S-acylation is an essential post-translational modification, which is mediated by a family of 23 zDHHC enzymes in humans. Several thousand proteins are modified by S-acylation; however, we lack a detailed understanding of how enzyme–substrate recognition and specificity is achieved. Previous work showed that the ankyrin repeat domain of zDHHC17 (ANK17) recognizes a short linear motif, known as the zDHHC ANK binding motif (zDABM) in substrate protein SNAP25, as a mechanism of substrate recruitment prior to S-acylation. Here, we investigated the S-acylation of the Sprouty and SPRED family of proteins by zDHHC17. Interestingly, although Sprouty-2 (Spry2) contains a zDABM that interacts with ANK17, this mode of binding is dispensable for S-acylation, and indeed removal of the zDABM does not completely ablate binding to zDHHC17. Furthermore, the related SPRED3 protein interacts with and is efficiently S-acylated by zDHHC17, despite lacking a zDABM. We undertook mutational analysis of SPRED3 to better understand the basis of its zDABM-independent interaction with zDHHC17. This analysis found that the cysteine-rich SPR domain of SPRED3, which is the defining feature of all Sprouty and SPRED proteins, interacts with zDHHC17. Surprisingly, the interaction with SPRED3 was independent of ANK17. Our mutational analysis of Spry2 was consistent with the SPR domain of this protein containing a zDHHC17-binding site, and Spry2 also showed detectable binding to a zDHHC17 mutant lacking the ANK domain. Thus, zDHHC17 can recognize its substrates through zDABM-dependent and/or zDABM–independent mechanisms, and some substrates display more than one mode of binding to this enzyme.

S-酰化是一种重要的翻译后修饰，由人类中的 23 种 zDHHC 酶家族介导。数千种蛋白质通过 S-酰化进行修饰；然而，我们对酶-底物识别和特异性是如何实现的缺乏详细的了解。先前的工作表明，zDHHC17 (ANK17) 的锚蛋白重复结构域识别底物蛋白 SNAP25 中的一个短线性基序，称为 zDHHC ANK 结合基序 (zDABM)，作为 S-酰化之前的底物招募机制。在这里，我们研究了 zDHHC17 对 Sprouty 和 SPRED 蛋白家族的 S 酰化。有趣的是，虽然 Sprouty-2 (Spry2) 含有与 ANK17 相互作用的 zDABM，但这种结合模式对于 S-酰化来说是可有可无的，而且事实上，去除 zDABM 并不能完全消除与 zDHHC17 的结合。此外，相关的 SPRED3 蛋白与 zDHHC17 相互作用并被 zDHHC17 有效地 S 酰化，尽管缺乏 zDABM。我们对 SPRED3 进行了突变分析，以更好地了解其与 zDHHC17 独立于 zDABM 的相互作用的基础。该分析发现，SPRED3 富含半胱氨酸的 SPR 结构域（所有 Sprouty 和 SPRED 蛋白的定义特征）与 zDHHC17 相互作用。令人惊讶的是，与 SPRED3 的相互作用独立于 ANK17。我们对 Spry2 的突变分析与该蛋白含有 zDHHC17 结合位点的 SPR 结构域一致，并且 Spry2 还显示出与缺乏 ANK 结构域的 zDHHC17 突变体的可检测结合。因此，zDHHC17 可以通过 zDABM 依赖性和/或 zDABM 独立机制识别其底物，并且一些底物表现出不止一种与该酶结合的模式。