INTRODUCTION: The Phox (PX) domain is a 125 residue module found in at least 167 eukaryotic proteins including kinesins, phospholipases, phosphoinositide (PI) 3-kinases, protein kinases, SNAREs and sorting nexins. It is named for its presence in NADPH oxidase subunits which, when mutated, cause chronic granulomatous disease. Most proteins which contain PX domains are involved in membrane trafficking, actin cytoskeletal organization and protein sorting. However, the ligand and structure of the PX domain are unknown. We have selected the PX domain of Vam7 as the focus of our project. Vam7 is a t-SNARE that contains a N-terminal PX domain and a C-terminal coiled coil, but unlike most SNAREs, lacks a known membrane interaction domain. Nonetheless, Vam7 is targeted to vacuolar membranes where it acts as a SNAP-25 homolog in the docking and fusion of cargo vesicles from the Golgi apparatus. We have demonstrated that Vam7's PX domain targets the SNARE complex that fuses vacuolar and vesicular membranes for cargo delivery (in press). We have shown that the PX domain is a novel signaling module that specifically targets PtdIns(3)P, and now seek to establish its structural mechanism of action. OBJECTIVES:
We aim to solve the solution structures of a PX domain in several functional states to define the mechanism of membrane targeting by this signaling module. Solution structures of the PtdIns(3)P-bound and micelle-bound states will be elucidated by heteronuclear magnetic resonance spectroscopy. Interactions with a variety of micelles and membrane mimetics will be explored. The structures and binding properties of the PX domain will be compared to other PI recognition modules such as FYVE, ENTH and pleckstrin homology (PH) domains to elucidate common membrane recognition principles.
Our specific aims are to (1) solve the 3D structure of the PX domain in the free state, (2) solve the structure of the complex with PtdIns(3)P, and (3) elucidate the structural basis of the PX domain's interaction with micelles or membranes.
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| Lumb, Craig N; He, Ju; Xue, Yi et al. (2011) Biophysical and computational studies of membrane penetration by the GRP1 pleckstrin homology domain. Structure 19:1338-46 |
| He, Ju; Kutateladze, Tatiana G (2008) Sequence-specific 1H, 13C, and 15N resonance assignments of GRP1 PH domain. Biomol NMR Assign 2:97-9 |
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