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X-ray crystal structure of CMS1MS2: a high proteolytic activity cysteine proteinase from Carica candamarcensis

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Abstract

CMS1MS2 (CC-Ib) from Carica candamarcensis (Vasconcellea cundinamarcensis) is a cysteine proteinase found as a single polypeptide containing 213 residues of 22,991 Da. The enzyme was purified by three chromatographic steps, two of them involving cationic exchange. Crystals of CMS1MS2 complexed with E-64 were obtained by the hanging drop vapor-diffusion method at 291 K using ammonium sulfate and polyethylene glycol 4000/8000 as precipitant. The complex CMS1MS2-E-64 crystallized in the tetragonal space group P41212 with unit-cell parameters; a = b = 73.64, c = 118.79 Å. The structure was determined by Molecular Replacement and refined at 1.87 Å resolution to a final R factor of 16.2 % (R free = 19.3 %). Based on the model, the structure of CMS1MS2 (PDB 3IOQ) ranks as one of the least basic cysteine isoforms from C. candamarcensis, is structurally closer to papain, caricain, chymopapain and mexicain than to the other cysteine proteinases, while its activity is twice the activity of papain towards BAPNA substrate. Two differences, one in the S2 subsite and another in the S3 subsite of CMS1MS2 may contribute to the enhanced activity relative to papain. In addition, the model provides a structural basis for the sensitivity of CMS1MS2 to inhibition by cystatin, not shown by other enzymes of the group, e.g., glycyl endopeptidase and CMS2MS2.

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Acknowledgments

The research was funded by CNPq, CAPES and Fapemig. We thank Dr. Abraham Schnaiderman for his financial aid to this research and the Brazilian Synchrotron Light Laboratory (LNLS) for providing access to their facilities for X-ray crystallography experiments. Each author confirms that there is no conflict of interest in connection with this manuscript.

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  1. Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG, 31270-901, Brazil

    Marco T. R. Gomes, Raphael D. Teixeira, Ronaldo A. P. Nagem & Carlos E. Salas

  2. Departamento de Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG, 31270-901, Brazil

    Míriam T. P. Lopes

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  1. Marco T. R. Gomes
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Correspondence to Carlos E. Salas.

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Gomes, M.T.R., Teixeira, R.D., Lopes, M.T.P. et al. X-ray crystal structure of CMS1MS2: a high proteolytic activity cysteine proteinase from Carica candamarcensis . Amino Acids 43, 2381–2391 (2012). https://doi.org/10.1007/s00726-012-1318-7

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