Solution structure of the Vam7p PX domain.
PX domains have been recently found to act as phosphoinositide binding modules. In the yeast SNARE protein Vam7p, the PX domain binds to PtdIns(3)P and is required for vacuolar targeting. To gain insight into how PX domains function, the solution structure of the ligand-free Vam7p PX domain has been determined by NMR spectroscopy. The Vam7p PX domain has the same overall alpha/beta fold observed in the structures of the ligand-free p47(phox) PX domain and the PtdIns(3)P-bound p40(phox) PX domain, exhibiting several similarities and differences with these two PX domains. Most striking is the similarity between the Vam7p and p40(phox) PX domains in a subset of secondary structure elements despite the low level of sequence identity between them, suggesting that these elements form a conserved core in the PX domain fold. These similarities and the observation that a putative PtdIns(3)P binding site is already formed in the apo Vam7p PX domains suggest that ligand binding does not induce major conformational changes, contrary to what was previously thought. The proposed ligand binding site of the Vam7p PX domain includes basic side chains from the conserved structural core that also participate in PtdIns(3)P binding to the p40(phox) PX domain, and basic side chains from a variable loop that probably inserts into the membrane. These results indicate that PX domains contain a combination of conserved and variable features that allow them to have a common function and at the same time exhibit distinct specificities, mechanisms of regulation, or modes of interaction with effector molecules.
Lu
Jun
J
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
Garcia
Jesus
J
Dulubova
Irina
I
Südhof
Thomas C
TC
Rizo
Josep
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019255:NADPH Oxidases; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D050825:Synaptosomal-Associated Protein 25; D013816:Thermodynamics
D005656:Fungal Proteins; D008024:Ligands; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012996:Solutions; D050825:Synaptosomal-Associated Protein 25; C085072:neutrophil cytosol factor 40K; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1