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 Ca²⁺-binding protein and interacts with a variety of intracellular proteins. Two types of ALG-2-binding motifs have been determined: type 1, P<em>X</em>YP<em>X</em>nYP (<em>X</em>, variable; <em>n</em> = 4), in ALIX and PLSCR3; type 2, P<em>X</em>PGF, 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 <em>N</em>-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-<em>S</em>-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.

Keywords

• ALG-2
• calcium-binding protein
• computational prediction
• protein-protein interaction
• proline-rich motif