Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors

Marie Sofie Møller, Malene Bech Vester-Christensen, Johanne M. Jensen, Maher Abou Hachem, Anette Henriksen, Birte Svensson

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    Abstract

    Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.
    Original languageEnglish
    JournalJournal of Biological Chemistry
    Volume290
    Issue number20
    Pages (from-to)12614-12629
    ISSN0021-9258
    DOIs
    Publication statusPublished - 2015

    Keywords

    • Crystal structure
    • Enzyme inhibitor
    • Plant molecular biology
    • Protein complex
    • Protein-protein interaction
    • Barley limit dextrinase
    • Cereal type inhibitors
    • Debranching enzyme
    • Seed germination
    • Starch metabolism regulation

    Cite this

    @article{aeae2cd7cbe54d0db08c57b244f28f3f,
    title = "Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors",
    abstract = "Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 {\AA}. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.",
    keywords = "Crystal structure, Enzyme inhibitor, Plant molecular biology, Protein complex, Protein-protein interaction, Barley limit dextrinase, Cereal type inhibitors, Debranching enzyme, Seed germination, Starch metabolism regulation",
    author = "M{\o}ller, {Marie Sofie} and Vester-Christensen, {Malene Bech} and Jensen, {Johanne M.} and {Abou Hachem}, Maher and Anette Henriksen and Birte Svensson",
    year = "2015",
    doi = "10.1074/jbc.M115.642777",
    language = "English",
    volume = "290",
    pages = "12614--12629",
    journal = "Journal of Biological Chemistry",
    issn = "0021-9258",
    publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
    number = "20",

    }

    Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors. / Møller, Marie Sofie; Vester-Christensen, Malene Bech; Jensen, Johanne M.; Abou Hachem, Maher ; Henriksen, Anette; Svensson, Birte.

    In: Journal of Biological Chemistry, Vol. 290, No. 20, 2015, p. 12614-12629.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors

    AU - Møller, Marie Sofie

    AU - Vester-Christensen, Malene Bech

    AU - Jensen, Johanne M.

    AU - Abou Hachem, Maher

    AU - Henriksen, Anette

    AU - Svensson, Birte

    PY - 2015

    Y1 - 2015

    N2 - Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.

    AB - Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.

    KW - Crystal structure

    KW - Enzyme inhibitor

    KW - Plant molecular biology

    KW - Protein complex

    KW - Protein-protein interaction

    KW - Barley limit dextrinase

    KW - Cereal type inhibitors

    KW - Debranching enzyme

    KW - Seed germination

    KW - Starch metabolism regulation

    U2 - 10.1074/jbc.M115.642777

    DO - 10.1074/jbc.M115.642777

    M3 - Journal article

    VL - 290

    SP - 12614

    EP - 12629

    JO - Journal of Biological Chemistry

    JF - Journal of Biological Chemistry

    SN - 0021-9258

    IS - 20

    ER -