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Int. J. Mol. Sci. 2015, 16(2), 3677-3699; doi:10.3390/ijms16023677

Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2

1
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
2
Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japan
3
Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8020, New Zealand
4
Department of Structural Biology, School of Medicine, Stanford University, Stanford, CA 94305-5126, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Charles A. Collyer
Received: 9 January 2015 / Accepted: 30 January 2015 / Published: 6 February 2015
(This article belongs to the Special Issue Protein Crystallography in Molecular Biology 2015)
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Abstract

ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca2+-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe85, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr180, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined. View Full-Text
Keywords: adaptor protein; calcium-binding protein; COPII; crystal structure; EF-hand; motif; protein-protein interaction adaptor protein; calcium-binding protein; COPII; crystal structure; EF-hand; motif; protein-protein interaction
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Takahashi, T.; Kojima, K.; Zhang, W.; Sasaki, K.; Ito, M.; Suzuki, H.; Kawasaki, M.; Wakatsuki, S.; Takahara, T.; Shibata, H.; Maki, M. Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2. Int. J. Mol. Sci. 2015, 16, 3677-3699.

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