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Int. J. Mol. Sci. 2012, 13(6), 7532-7549; doi:10.3390/ijms13067532
Article

Prediction of a New Ligand-Binding Site for Type 2 Motif based on the Crystal Structure of ALG-2 by Dry and Wet Approaches

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Received: 5 April 2012; in revised form: 6 June 2012 / Accepted: 13 June 2012 / Published: 18 June 2012
(This article belongs to the Section Molecular Recognition)
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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, 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.
Keywords: ALG-2; calcium-binding protein; computational prediction; protein-protein interaction; proline-rich motif ALG-2; calcium-binding protein; computational prediction; protein-protein interaction; proline-rich motif
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Takahashi, T.; Suzuki, H.; Inuzuka, T.; Shibata, H.; Maki, M. Prediction of a New Ligand-Binding Site for Type 2 Motif based on the Crystal Structure of ALG-2 by Dry and Wet Approaches. Int. J. Mol. Sci. 2012, 13, 7532-7549.

AMA Style

Takahashi T, Suzuki H, Inuzuka T, Shibata H, Maki M. Prediction of a New Ligand-Binding Site for Type 2 Motif based on the Crystal Structure of ALG-2 by Dry and Wet Approaches. International Journal of Molecular Sciences. 2012; 13(6):7532-7549.

Chicago/Turabian Style

Takahashi, Takeshi; Suzuki, Hironori; Inuzuka, Tatsutoshi; Shibata, Hideki; Maki, Masatoshi. 2012. "Prediction of a New Ligand-Binding Site for Type 2 Motif based on the Crystal Structure of ALG-2 by Dry and Wet Approaches." Int. J. Mol. Sci. 13, no. 6: 7532-7549.



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