Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29

Funder: Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (RES0043758, RES0046091)Abstract: VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn++ microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established

Structure/Function Studies on Vps9 domain-containing Ankyrin Repeat protein (VARP)

Vps-9 domain ankyrin repeat protein (Varp) is a widely expressed multi- domain protein of 1050 amino acids that resides in the cytosol. Varp has been shown to be a Rab21 GEF, a Rab32/38 effector and also helps to regulate VAMP7 fusion activity. Varp has more recently been proposed to bind to the Golgi tether golginA4, the kinesin motor Kif5a and the retromer complex subunit Vps29. The retromer core complex, comprising Vps35, Vps26, and Vps29, utilises different SNX proteins to mediate both Endosome to Golgi traffic, and Endosome to Plasma membrane traffic. The interaction of Varp with Vps29 localises Varp to endosomal membranes. Varp has two ‘zinc knuckle’ sequences (residues 369- 460 and 692-746) that have been shown to bind zinc and it is these small sequences that mediate the interaction with Vps29. The Varp:Vps29 complex structure was determined by a combined X-Ray Crystallographic/NMR method. This thesis demonstrates that one zinc knuckle sequence of Varp is sufficient to form an interaction with Vps29, with an affinity in the micromolar range. Single point mutations in Vps29 are sufficient to affect the binding affinity of Varp. Additionally, as some reported functional interactions have not yet been localised to any particular region of Varp, the as- yet-unstudied N-terminal domain of Varp (residues 1-136) was investigated using a Yeast-2-Hybrid screening approach, to try to identify any interaction partners that bind in this region. Many potential interaction partners were discovered, however neither Kif5a nor golginA4 was detected binding in this region

Measuring protein reduction potentials using 15N HSQC NMR spectroscopy

NMR spectroscopy was used to measure reduction potentials of four redox proteins by following multiple 15N HSQC protein resonances across a titration series using mixtures of oxidised and reduced glutathione. Results for PDI a, PDI ab and DsbA agree with the literature and our result for ERp18 confirms this protein as an oxidoreductase of comparable or greater reducing strength than PDI a

Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29

Funder: Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (RES0043758, RES0046091)Abstract: VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn++ microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established