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 Feb 6;16(2):3677-99. doi: 10.3390/ijms16023677.

Takeshi Takahashi ¹, Kyosuke Kojima ², Wei Zhang ³, Kanae Sasaki ⁴, Masaru Ito ⁵, Hironori Suzuki ⁶ ⁷, Masato Kawasaki ⁸, Soichi Wakatsuki ⁹ ¹⁰, Terunao Takahara ¹¹, Hideki Shibata ¹², Masatoshi Maki ¹³

Affiliations

1 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. takahashi.takeshi@a.mbox.nagoya-u.ac.jp.
2 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. kojima.kiyousuke@c.mbox.nagoya-u.ac.jp.
3 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. zhang.wei@i.mbox.nagoya-u.ac.jp.
4 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. guinaijiang@hotmail.co.jp.
5 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. kurukurumegane3310@gmail.com.
6 Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japan. hironori.suzuki@canterbury.ac.nz.
7 Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8020, New Zealand. hironori.suzuki@canterbury.ac.nz.
8 Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japan. masato.kawasaki@kek.jp.
9 Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japan. soichi.wakatsuki@stanford.edu.
10 Department of Structural Biology, School of Medicine, Stanford University, Stanford, CA 94305-5126, USA. soichi.wakatsuki@stanford.edu.
11 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. takahara@agr.nagoya-u.ac.jp.
12 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. shibabou@agr.nagoya-u.ac.jp.
13 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. mmaki@agr.nagoya-u.ac.jp.

PMID: 25667979 DOI: 10.3390/ijms16023677

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.

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