title: Prediction of a new ligand-binding site for type 2 motif based on the crystal structure of ALG-2 by dry and wet approaches.
abstract: ALG-2 is a penta-EF-hand Ca(2+)-binding protein and interacts with a variety of intracellular proteins. Two types of ALG-2-binding motifs have been determined: type 1, PXYPXnYP (X, variable; n = 4), in ALIX and PLSCR3; type 2, PXPGF, in Sec31A and PLSCR3. The previously solved X-ray crystal structure of the complex between ALG-2 and an ALIX peptide containing type 1 motif showed that the peptide binds to Pocket 1 and Pocket 2. Co-crystallization of ALG-2 and type 2 motif-containing peptides has not been successful. To gain insights into the molecular basis of type 2 motif recognition, we searched for a new hydrophobic cavity by computational algorithms using MetaPocket 2.0 based on 3D structures of ALG-2. The predicted hydrophobic pocket designated Pocket 3 fits with N-acetyl-ProAlaProGlyPhe-amide, a virtual penta-peptide derived from one of the two types of ALG-2-binding sites in PLSCR3 (type 2 motif), using the molecular docking software AutoDock Vina. We investigated effects of amino acid substitutions of the predicted binding sites on binding abilities by pulldown assays using glutathione-S-transferase -fused ALG-2 of wild-type and mutant proteins and lysates of cells expressing green fluorescent protein -fused PLSCR3 of wild-type and mutants. Substitution of either L52 with Ala or F148 with Ser of ALG-2 caused loss of binding abilities to PLSCR3 lacking type 1 motif but retained those to PLSCR3 lacking type 2 motif, strongly supporting the hypothesis that Pocket 3 is the binding site for type 2 motif.
authors: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors: Takeshi
authors: T
authors: Takahashi
authors: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors: Hironori
authors: H
authors: Suzuki
authors: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors: Tatsutoshi
authors: T
authors: Inuzuka
authors: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors: Hideki
authors: H
authors: Shibata
authors: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors: Masatoshi
authors: M
authors: Maki
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D008024:Ligands; D051379:Mice; D020125:Mutation, Missense
chemical_list: D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D008024:Ligands; C490815:Pdcd6 protein, mouse