The Habc domain of syntaxin 3 is a ubiquitin binding domain

Adrian J Giovannone,Rashid Syed,Sirpi Nackeeran,Elena Reales,Pallavi Bhattaram,Adam B Monahan,Thomas Weimbs

doi:10.1038/s41598-020-78412-0

Abstract

Syntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in eukaryotic intracellular membrane trafficking pathways. Syntaxins contain an N-terminal regulatory domain, termed the Habc domain that is not highly conserved at the primary sequence level but folds into a three-helix bundle that is structurally conserved among family members. The syntaxin Habc domain has previously been found to be structurally very similar to the GAT domain present in GGA family members and related proteins that are otherwise completely unrelated to syntaxins. Because the GAT domain has been found to be a ubiquitin binding domain we hypothesized that the Habc domain of syntaxins may also bind to ubiquitin. Here, we report that the Habc domain of syntaxin 3 (Stx3) indeed binds to monomeric ubiquitin with low affinity. This domain binds efficiently to K63-linked poly-ubiquitin chains within a narrow range of chain lengths but not to K48-linked poly-ubiquitin chains. Other syntaxin family members also bind to K63-linked poly-ubiquitin chains but with different chain length specificities. Molecular modeling suggests that residues of the GGA3-GAT domain known to be important for ionic and hydrophobic interactions with ubiquitin may have equivalent, conserved residues within the Habc domain of Stx3. We conclude that the syntaxin Habc domain and the GAT domain are both structurally and functionally related, and likely share a common ancestry despite sequence divergence. Binding of Ubiquitin to the Habc domain may regulate the function of syntaxins in membrane fusion or may suggest additional functions of this protein family.

Highlights

Syntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in eukaryotic intracellular membrane trafficking pathways
We have recently reported that syntaxin 3, a SNARE involved in membrane fusion at the apical plasma membrane of polarized epithelial cells, undergoes mono-ubiquitination at lysine residues adjacent to its transmembrane domain[34]
A GST-fusion protein containing only the H abc domain (1–146) of Stx[3] pulls down with ubiquitin-coated beads much more efficiently than the entire Stx[3] cytoplasmic domain (3.78 fold change increase) (Fig. 1c). This suggests that the H abc domain directly binds to ubiquitin, and that this interaction is inhibited by intramolecular binding between the H abc and SNARE domains in the “closed conformation” of Stx[3]

Summary

Introduction

Syntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in eukaryotic intracellular membrane trafficking pathways. Syntaxins contain an N-terminal regulatory domain, termed the Habc domain that is not highly conserved at the primary sequence level but folds into a three-helix bundle that is structurally conserved among family members. In the cases of syntaxin family members whose Habc domains have been structurally studied it was found that they all share a highly conserved fold This includes Stx1A10,11, Stx612, Sso113, Stx1014, Vam3p15 and several others (see Protein Data Bank) whose H abc domains fold into essentially superimposable three-helix bundles despite limited or absent sequence similarity. In some—but not all—syntaxins the H abc domains have the ability to engage in an intramolecular interaction with the SNARE domain resulting in a tetrameric helical bundle This “closed” conformation generally inhibits the formation of complexes with cognate SNARE proteins and thereby inhibits membrane fusion[16,17]. The GAT domains of GGA proteins were found to be ubiquitin binding domains[19,20,21,22,23,24] which helped to explain their function in recruiting ubiquitinated membrane proteins for targeting to multivesicular bodies (MVBs)[25]

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