X-ray crystallography and NMR studies of domain-swapped canecystatin-1

Authors

  • Napoleão F. Valadares,

    1. Center for Structural Molecular Biotechnology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, Brazil
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  • Rodrigo de Oliveira-Silva,

    1. Center for Structural Molecular Biotechnology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, Brazil
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  • Italo A. Cavini,

    1. Center for Structural Molecular Biotechnology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, Brazil
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  • Ivo de Almeida Marques,

    1. Physics Institute, Federal University of Goiás, Brazil
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  • Humberto D'Muniz Pereira,

    1. Center for Structural Molecular Biotechnology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, Brazil
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  • Andrea Soares-Costa,

    1. Laboratory of Molecular Biology, Department of Genetic and Evolution, Federal University of São Carlos, Brazil
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  • Flavio Henrique-Silva,

    1. Laboratory of Molecular Biology, Department of Genetic and Evolution, Federal University of São Carlos, Brazil
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  • Hans R. Kalbitzer,

    1. Institute of Biophysics and Physical Biochemistry, University of Regensburg, Germany
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  • Claudia E. Munte,

    1. Center for Structural Molecular Biotechnology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, Brazil
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  • Richard C. Garratt

    Corresponding author
    • Center for Structural Molecular Biotechnology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, Brazil
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Correspondence

R. C. Garratt, Center for Structural Molecular Biotechnology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, Av. Trabalhador são-carlense 400, São Carlos-SP 13566-570, Brazil

Fax: +55 16 3373 9881

Tel: +55 16 3373-9881

E-mail: richard@ifsc.usp.br

Abstract

The three-dimensional structure of canecystatin-1, a potent inhibitor of cysteine proteases from sugarcane (Saccharum officinarum), has been solved in two different crystal forms. In both cases, it is seen to exist as a domain-swapped dimer, the first such observation for a cystatin of plant origin. Size exclusion chromatography and multidimensional NMR spectroscopy show the dimer to be the dominant species in solution, despite the presence of a measurable quantity of monomer undergoing slow exchange. The latter is believed to be the active species, whereas the domain-swapped dimer is presumably inactive, as its first inhibitory loop has been extended to form part of a long β-strand that forms a double-helical coiled coil with its partner from the other monomer. A similar structure is observed in human cystatin C, but the spatial disposition of the two lobes of the dimer is rather different. Dimerization is presumably a mechanism by which canecystatin-1 can be kept inactive within the plant, avoiding the inhibition of endogenous proteases. The structure described here provides a platform for the rational design of specific cysteine protease inhibitors for biotechnological applications.

Database

The coordinates and structure factors have been deposited in the Protein Data Bank under the accession codes 3UL5 and 3UL6.

Structured digital abstract

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