Structural studies of sorting nexin proteins from two distinct subfamilies

Abstract

Sorting nexin (SNX) proteins are a large family of proteins with critical roles in endocytosis, membrane trafficking and intracellular signalling. Each SNX protein contains a Phox homology (PX) domain that typically recognizes phosphoinositide lipids to enable their anchoring to defined organelles. SNXs also contain additional domains other than the PX domain thus allowing them to participate in a wide range of functions such as proteinprotein interactions, membrane remodeling, lipid metabolism and other functions that are yet to be explored. My research focuses on the structural and functional characterization of two different sub-families of SNX proteins; those that have both PX and BAR (bin/amphiphysin/rvs) domains (SNX5, SNX6 and SNX32), and the poorly characterized SNX-RGS sub-family that contain a regulator of G-protein signaling (RGS) domain (SNX13, SNX14, SNX19 and SNX25). These protein families are required for membrane trafficking and lipid droplet formation, and are implicated in diseases including pathogen invasion and cerebellar ataxia respectively. I first describe the structural mechanism for how a Chlamydial effector protein called IncE hijacks and recruits SNX5 related proteins to the bacterial inclusion membrane during Chlamydial infection (Chapter 2). Using X-ray crystallography, I demonstrate that the Cterminal region of IncE forms a β-hairpin structure and binds to a unique α-helical insertion on the PX domain of SNX5, which is a separate site from the canonical PX lipid-binding site. Using isothermal titration calorimetry, I also investigate the binding mechanisms of the SNX5 homologs, SNX6 and SNX32 to IncE, and demonstrate that these three proteins share a common binding mechanism. These results suggest that the IncE protein mimics other SNX5-related proteins, where SNX5 PX might be functioning as a protein-binding scaffold that could potentially orchestrate the trafficking of certain transmembrane receptors.The research presented in Chapter 3 extends the work described in Chapter 2 confirming the IncE binding site in the SNX32 PX domain using X-ray crystallography. The crystal structure of SNX32 PX-IncE complex not only confirmed the biophysical data from chapter 2, but also revealed the binding mechanism to be identical to the SNX5PX-IncE binding. In addition, this is the first reported structure of the SNX32 protein in either an apo or ligand bound state. This result further strengthens the idea that SNX-BAR proteins can act as receptor recyclers in the cell.In Chapter 4 using biophysical and structural studies, I next investigated the potential functional role of the SNX-BAR proteins in transmembrane cargo trafficking. My biophysical experiments directly confirmed the recent indirect proteomic studies that Conference abstractsStructural basis for the hijacking of endosomal sorting nexin proteins by Chlamydia trachomatis.