Crystal structure of greglin, a novel non-classical Kazal inhibitor, in complex with subtilisin

Authors

  • Chrystelle Derache,

    1. Centre de Biophysique Moléculaire, UPR 4301 CNRS conventionnée avec l'Université d'Orléans, Orléans Cedex 2, France
    Current affiliation:
    1. Medical Microbiology, Department of Laboratory Medicine Malmö, Lund University, Skåne University Hospital, Malmö, Sweden
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  • Christophe Epinette,

    1. Pathologies Respiratoires, Protéolyse et Aérosolthérapie, INSERM U1100 Faculté de Médecine, Université François Rabelais, Tours, France
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  • Alain Roussel,

    1. Architecture et Fonction des Macromolécules Biologiques, CNRS UMR7257 and Aix-Marseille Université, Marseille Cedex, France
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  • Guillaume Gabant,

    1. Centre de Biophysique Moléculaire, UPR 4301 CNRS conventionnée avec l'Université d'Orléans, Orléans Cedex 2, France
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  • Martine Cadene,

    1. Centre de Biophysique Moléculaire, UPR 4301 CNRS conventionnée avec l'Université d'Orléans, Orléans Cedex 2, France
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  • Brice Korkmaz,

    1. Pathologies Respiratoires, Protéolyse et Aérosolthérapie, INSERM U1100 Faculté de Médecine, Université François Rabelais, Tours, France
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  • Francis Gauthier,

    1. Pathologies Respiratoires, Protéolyse et Aérosolthérapie, INSERM U1100 Faculté de Médecine, Université François Rabelais, Tours, France
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  • Christine Kellenberger

    Corresponding author
    • Architecture et Fonction des Macromolécules Biologiques, CNRS UMR7257 and Aix-Marseille Université, Marseille Cedex, France
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Correspondence

C. Kellenberger, Architecture et Fonction des Macromolécules Biologiques, CNRS UMR7257 and Aix-Marseille Université, 13288 Marseille Cedex, France

Fax: +33 4 91 26 67 20

Tel: +33 4 91 82 55 83

E-mail: christine.kellenberger@afmb.univ-mrs.fr

Abstract

Greglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively; it also inhibits neutrophil cathepsin G, α-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95 °C) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1–20 and 21–83. Mass spectrometry indicates that the N-terminal domain (1–20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21–78 in complex with subtilisin was determined at 1.75 Å resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70–83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity.

Database

The atomic coordinates and structure factors for the greglin–subtilisin complex have been deposited with the RCSB Protein Data Bank under accession number 4GI3.

Structured digital abstract

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