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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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Homology model of clone A10 showing the residues correspondent to the point mutations. Interactions of (A) threonine 30 (magenta) and (B) glutamine 97 (orange). Interacting residues are labeled and shown as spheres. It is likely that the mutations would significantly destabilize the hydrophobic core leading to a conformation different to that shown in the figure.
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Figure 4: Homology model of clone A10 showing the residues correspondent to the point mutations. Interactions of (A) threonine 30 (magenta) and (B) glutamine 97 (orange). Interacting residues are labeled and shown as spheres. It is likely that the mutations would significantly destabilize the hydrophobic core leading to a conformation different to that shown in the figure.

Mentions: As depicted in Figure 4A, the first mutation (I30T) is located at the beginning of the α-helix where it would be expected to destabilize the hydrophobic cluster formed by residues F50, L53, I30 the aliphatic portion of R34 and the loop connecting the N-terminus to the α-helix (Additional file 2). The second mutation (L97Q) appears yet more significant and perturbs the opposite side of the hydrophobic core formed by L32, F35, A36, V86, F100 and L97 (Figure 4B). We suggest that these mutations would significantly destabilize the hydrophobic contacts which hold the helix against the β-sheet, thus leading to its complete or partial release. This release would have the effect of decoupling two components of the inhibitor active site; the N-terminal region on the one hand and the remaining two loops on the other.


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)

Homology model of clone A10 showing the residues correspondent to the point mutations. Interactions of (A) threonine 30 (magenta) and (B) glutamine 97 (orange). Interacting residues are labeled and shown as spheres. It is likely that the mutations would significantly destabilize the hydrophobic core leading to a conformation different to that shown in the figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Homology model of clone A10 showing the residues correspondent to the point mutations. Interactions of (A) threonine 30 (magenta) and (B) glutamine 97 (orange). Interacting residues are labeled and shown as spheres. It is likely that the mutations would significantly destabilize the hydrophobic core leading to a conformation different to that shown in the figure.
Mentions: As depicted in Figure 4A, the first mutation (I30T) is located at the beginning of the α-helix where it would be expected to destabilize the hydrophobic cluster formed by residues F50, L53, I30 the aliphatic portion of R34 and the loop connecting the N-terminus to the α-helix (Additional file 2). The second mutation (L97Q) appears yet more significant and perturbs the opposite side of the hydrophobic core formed by L32, F35, A36, V86, F100 and L97 (Figure 4B). We suggest that these mutations would significantly destabilize the hydrophobic contacts which hold the helix against the β-sheet, thus leading to its complete or partial release. This release would have the effect of decoupling two components of the inhibitor active site; the N-terminal region on the one hand and the remaining two loops on the other.

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