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

Valadares NF, Dellamano M, Soares-Costa A, Henrique-Silva F, Garratt RC - BMC Struct. Biol. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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.

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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Sequence alignment of relevant cystatins. Sequence alignment between oryzacystain-1 (cyan), canecystatin-1 (green), A10 and canecystatin-4. Conserved amino acids are show in red. Residues conserved in all sequences except canecystatin-4 are coloured in yellow. In the case of the clone A10 the residues are shaded according to the cystatin from which they were derived with point mutations shaded in purple, and the N-terminal deletion from residues 11 to 19 is shown as a blue gap. The clone OC-I NΔ has the sequence of oryzacystatin-1, but with the same N-terminal deletion as the clone A10.
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Figure 2: Sequence alignment of relevant cystatins. Sequence alignment between oryzacystain-1 (cyan), canecystatin-1 (green), A10 and canecystatin-4. Conserved amino acids are show in red. Residues conserved in all sequences except canecystatin-4 are coloured in yellow. In the case of the clone A10 the residues are shaded according to the cystatin from which they were derived with point mutations shaded in purple, and the N-terminal deletion from residues 11 to 19 is shown as a blue gap. The clone OC-I NΔ has the sequence of oryzacystatin-1, but with the same N-terminal deletion as the clone A10.

Mentions: Four differences can be noted between A10 and the original canecystatin-1 from which it is largely derived. Firstly, the N-terminal region of A10 comes from oryzacystatin-1 and not canecystatin-1, a result of the gene shuffling process itself. Secondly, this region has suffered a 7 amino acid deletion (Figure 2). Finally, A10 has acquired two unexpected point mutations affecting hydrophobic residues of the protein core, I30T at the beginning of the α-helix and L97Q in strand β5 (residue numbers follow those of canecystatin-1 throughout the text unless otherwise stated, see Figure 2).


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

Valadares NF, Dellamano M, Soares-Costa A, Henrique-Silva F, Garratt RC - BMC Struct. Biol. (2010)

Sequence alignment of relevant cystatins. Sequence alignment between oryzacystain-1 (cyan), canecystatin-1 (green), A10 and canecystatin-4. Conserved amino acids are show in red. Residues conserved in all sequences except canecystatin-4 are coloured in yellow. In the case of the clone A10 the residues are shaded according to the cystatin from which they were derived with point mutations shaded in purple, and the N-terminal deletion from residues 11 to 19 is shown as a blue gap. The clone OC-I NΔ has the sequence of oryzacystatin-1, but with the same N-terminal deletion as the clone A10.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2959088&req=5

Figure 2: Sequence alignment of relevant cystatins. Sequence alignment between oryzacystain-1 (cyan), canecystatin-1 (green), A10 and canecystatin-4. Conserved amino acids are show in red. Residues conserved in all sequences except canecystatin-4 are coloured in yellow. In the case of the clone A10 the residues are shaded according to the cystatin from which they were derived with point mutations shaded in purple, and the N-terminal deletion from residues 11 to 19 is shown as a blue gap. The clone OC-I NΔ has the sequence of oryzacystatin-1, but with the same N-terminal deletion as the clone A10.
Mentions: Four differences can be noted between A10 and the original canecystatin-1 from which it is largely derived. Firstly, the N-terminal region of A10 comes from oryzacystatin-1 and not canecystatin-1, a result of the gene shuffling process itself. Secondly, this region has suffered a 7 amino acid deletion (Figure 2). Finally, A10 has acquired two unexpected point mutations affecting hydrophobic residues of the protein core, I30T at the beginning of the α-helix and L97Q in strand β5 (residue numbers follow those of canecystatin-1 throughout the text unless otherwise stated, see Figure 2).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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.

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus