Structural Predictions of the SNX-RGS Proteins Suggest They Belong to a New Class of Lipid Transfer Proteins.

Blessy Paul,Vikas A Tillu,W Mike Henne,Saroja Weeratunga,Brett M Collins,Hanaa Hariri

doi:10.3389/fcell.2022.826688

Abstract

Recent advances in protein structure prediction using machine learning such as AlphaFold2 and RosettaFold presage a revolution in structural biology. Genome-wide predictions of protein structures are providing unprecedented insights into their architecture and intradomain interactions, and applications have already progressed towards assessing protein complex formation. Here we present detailed analyses of the sorting nexin proteins that contain regulator of G-protein signalling domains (SNX-RGS proteins), providing a key example of the ability of AlphaFold2 to reveal novel structures with previously unsuspected biological functions. These large proteins are conserved in most eukaryotes and are known to associate with lipid droplets (LDs) and sites of LD-membrane contacts, with key roles in regulating lipid metabolism. They possess five domains, including an N-terminal transmembrane domain that anchors them to the endoplasmic reticulum, an RGS domain, a lipid interacting phox homology (PX) domain and two additional domains named the PXA and PXC domains of unknown structure and function. Here we report the crystal structure of the RGS domain of sorting nexin 25 (SNX25) and show that the AlphaFold2 prediction closely matches the experimental structure. Analysing the full-length SNX-RGS proteins across multiple homologues and species we find that the distant PXA and PXC domains in fact fold into a single unique structure that notably features a large and conserved hydrophobic pocket. The nature of this pocket strongly suggests a role in lipid or fatty acid binding, and we propose that these molecules represent a new class of conserved lipid transfer proteins.

Highlights

The sorting nexins (SNXs) are a large family of proteins with diverse structures and functions
The wildtype sequence of the regulator of G-protein signalling (RGS) domain crystallises with a non-native disulfide bond formed by Cys526 between adjacent monomers in the crystal lattice which distorts the orientation of the helix α5
We investigated how similar the experimentally determined sorting nexin 25 (SNX25) RGS domain was to the predicted structure from the AlphaFold2 database (Tunyasuvunakool et al, 2021)

Summary

Introduction

The sorting nexins (SNXs) are a large family of proteins with diverse structures and functions They are found in all eukaryotes and their defining feature is the presence of a phox homology (PX) domain, which most commonly associates with phosphatidylinositol phospholipids (PtdInsPs) to mediate interactions with membranes of the endolysosomal system (Teasdale and Collins, 2012; Chandra and Collins, 2019; Chandra et al, 2019). Other well-characterised family members include Mdm from Saccharomyces cerevisiae and Snazarus (Snz) from Drosophila melanogaster These proteins have relatively low sequence similarity across homologues and across species but share a conserved architecture with an N-terminal hydrophobic membrane anchor, and a central RGS domain and PX domain (except for SNX19 orthologues which lack the RGS domain). The N-terminal membrane anchor mediates localisation to the endoplasmic reticulum (ER) (Henne et al, 2015; Bryant et al, 2018; Datta et al, 2019; Ugrankar et al, 2019; Saric et al, 2021), and the proteins typically localise to sites of new lipid droplet synthesis and can enhance membrane tethering of the ER to other membrane compartments via their PX domains including the vacuole in yeast (Henne et al, 2015; Hariri et al, 2018; Hariri et al, 2019), the plasma membrane in Drosophila (Ugrankar et al, 2019) and endolysosomal compartments in mammalian cells (Bryant et al, 2018; Datta et al, 2019; Saric et al, 2021; Lu et al, 2022)

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