Open Access Research article

Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling

Napoleão F Valadares1, Márcia Dellamano2, Andrea Soares-Costa2, Flávio Henrique-Silva2* and Richard C Garratt1*

Author Affiliations

1 Center for Structural Molecular Biotechnology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, Av. Trabalhador são-carlense 400, 13560-970, São Carlos-SP, Brazil

2 Laboratory of Molecular Biology, Department of Genetic and Evolution, Federal University of São Carlos, Rodovia Washington Luis km 235, CEP 13565-905, São Carlos-SP, Brazil

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BMC Structural Biology 2010, 10:30  doi:10.1186/1472-6807-10-30

Published: 30 September 2010



Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis.


Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability.


A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.