Abstract
Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of the diverse PI specificity of PX domains, we determined the crystal structure of the PX domain from Bem1p that has been reported to bind phosphatidylinositol 4-phosphate (PtdIns(4)P). We also measured the membrane binding properties of the PX domain and its mutants by surface plasmon resonance and monolayer techniques and calculated the electrostatic potentials for the PX domain in the absence and presence of bound PtdIns(4)P. The Bem1p PX domain contains a signature PI-binding site optimized for PtdIns(4)P binding and also harbors basic and hydrophobic residues on the membrane-binding surface. The membrane binding of the Bem1p PX domain is initiated by nonspecific electrostatic interactions between the cationic membrane-binding surface of the domain and anionic membrane surfaces, followed by the membrane penetration of hydrophobic residues. Unlike other PX domains, the Bem1p PX domain has high intrinsic membrane penetrating activity in the absence of PtdIns(4)P, suggesting that the partial membrane penetration may occur before specific PtdIns(4)P binding and last after the removal of PtdIns(4)P under certain conditions. This structural and functional study of the PtdIns(4)P-binding Bem1p PX domain provides new insight into the diverse PI specificities and membrane-binding mechanisms of PX domains.
Highlights
Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities
Bem1p is a multidomain scaffolding protein that binds Cdc42p with its N-terminal Src homology 3 (SH3) domain (33), and this interaction is critical for proper Cdc42p activation (34)
Bem1p has been shown to migrate to the plasma inositol; PH, pleckstrin homology; Fab1/ YOTB/Vac1/EEA1 (FYVE), Fab1/YOTB/Vac1/EEA1; PX, Phox homology; ENTH, epsin N-terminal homology; SH3, Src homology 3; PLD, phospholipase D; CISK, cytokine-independent survival kinase; PI3K-conserved 2 (C2)␣, phosphoinositide 3-kinase C2␣; PS, phosphatidylserine; SPR, surface plasmon resonance; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MAD, multiwavelength anomalous dispersion; Bem1p-PX, Bem1p PX domain; OSBP, oxysterol-binding protein; PPII, type II polyproline helix
Summary
Materials—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were from Avanti Polar Lipids (Alabaster, AL). Phospholipid concentrations were determined by phosphate analysis (56). Fatty acid-free bovine serum albumin was from Bayer (Kankakee, IL). The protein was expressed in the methionine-requiring auxotrophic Escherichia coli strain 834(DE3) and purified by Ni2ϩ affinity, heparin, and gel filtration chromatography. The protein in gel filtration buffer (20 mM Tris-HCl (pH 7.4 at 25 °C), 100 mM NaCl, and 5 mM dithiothreitol) was concentrated to 5 mg/ml. Crystals were obtained in sitting drops (3 l of protein plus 3 l of reservoir solution) that were incubated at 14 °C over a reservoir consisting of 0.2 M NaCl, 0.1 M sodium/potassium phosphate (pH 6.2), 10% polyethylene glycol 8000, and 2 mM dithiothreitol.
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