title: Structural analysis of the complex between penta-EF-hand ALG-2 protein and Sec31A peptide reveals a novel target recognition mechanism of ALG-2.
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.
authors: 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.
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. kojima.kiyousuke@c.mbox.nagoya-u.ac.jp.
authors: Kyosuke
authors: K
authors: Kojima
authors: 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.
authors: Wei
authors: W
authors: Zhang
authors: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. guinaijiang@hotmail.co.jp.
authors: Kanae
authors: K
authors: Sasaki
authors: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. kurukurumegane3310@gmail.com.
authors: Masaru
authors: M
authors: Ito
authors: 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.|Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8020, New Zealand. hironori.suzuki@canterbury.ac.nz.
authors: Hironori
authors: H
authors: Suzuki
authors: 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.
authors: Masato
authors: M
authors: Kawasaki
authors: 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.|Department of Structural Biology, School of Medicine, Stanford University, Stanford, CA 94305-5126, USA. soichi.wakatsuki@stanford.edu.
authors: Soichi
authors: S
authors: Wakatsuki
authors: 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.
authors: Terunao
authors: T
authors: Takahara
authors: 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.
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. mmaki@agr.nagoya-u.ac.jp.
authors: Masatoshi
authors: M
authors: Maki
mesh_terms: D019943:Amino Acid Substitution; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics*
chemical_list: D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C097753:PDCD6 protein, human; C117020:PDCD6IP protein, human; D010455:Peptides; C507455:SEC31A protein, human; D033921:Vesicular Transport Proteins