Biophysical and structural characterization of the stress response protein CpxP

Abstract

Retromer is a peripheral membrane protein complex that plays a critical role in a broad range of physiological, developmental and pathological processes by mediating retrograde transport of proteins from endosomes to the trans-Golgi (TGN) network. Wnt signalling, toxin transport and amyloid production in Alzheimer's disease are just some of the processes known to be regulated by retromer-mediated trafficking. Mammalian retromer consists of a core heterotrimeric cargorecognition subcomplex associated with a membrane-targeting dimer of sorting nexins. The core subcomplex consists of vacuolar protein sorting (VPS)26, VPS29 and VPS35 subunits that play different roles in complex stabilisation and in binding to transport cargo and regulatory proteins. The composition of the membrane-binding subcomplex is inconclusive but consists of a homo-or heterodimer of sorting nexins, SNX1, SNX2, SNX5 and SNX6. These BAR (Bin/Amphiphysin/Rvs)domain proteins can induce the formation of high curvature membrane tubules through the formation of a polymerised helical coat. We have used small-angle X-ray scattering (SAXS), X-ray crystallography, nuclear magnetic resonance (NMR) and isothermal titration calorimetry to elucidate a qualitative and quantitative model of retromer assembly [1]. In our proposed model, VPS35 forms an extended, gently curved structure composed of alternating HEAT-like helical repeats. VPS26 and VPS29 bind to distal ends through N-and C-terminal regions of VPS35, respectively, to form a stable trimeric core assembly. Results from thermodynamics experiments have shown that VPS29 and VPS26 bind to VPS35 completely independently of each other, confirming that VPS35 plays the role of central scaffold and VPS29 and VPS26 do not form any contact with each other. Intriguingly, the core trimeric complex is able to form a symmetric dimer, which may have implications for functional interactions in vivo. Solution structures of SNX1 and SNX2 homodimers have been determined using SAXS. The SNX dimer associates with cellular membranes enriched in phosphatidylinositol 3-phosphate and 3,5diphosphate and is believed to polymerise into a helical protein coat. NMR studies have confirmed that VPS29 coordinates the binding of the core subcomplex to this membrane remodelling complex. This coupling of cargo binding and membrane tubulating events enable retromer to recruit cargo molecules into a tubular endosome-to-Golgi transport carrier.