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Interdomain Contacts and the Stability of Serralysin Protease from Serratia marcescens.

Zhang L, Morrison AJ, Thibodeau PH - PLoS ONE (2015)

Bottom Line: Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases.Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability.This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15219, United States of America.

ABSTRACT
The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX) family of toxins and virulence factors, which mediated virulence in E. coli, B. pertussis, and P. aeruginosa, as well as other animal and plant pathogens. The serralysin proteases are structurally dynamic and their folding is regulated by calcium binding to a C-terminal domain that defines the RTX family of proteins. Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases. Extending from this, stabilization of these interactions in the native structure may lead to hyperstabilization of the folded protein. To test this hypothesis, cysteine pairs were introduced into the N-terminal helix and the RTX domain and protease folding and activity were assessed. Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability. This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding. These data demonstrate that the thermal and chemical stability of these proteases is, in part, mediated by binding between the RTX domain and the N-terminal helix and demonstrate that stabilization of this interaction can further stabilize the active protease, leading to additional pH and thermal tolerance.

No MeSH data available.


Related in: MedlinePlus

Disulfide stabilization under relaxed biochemical conditions.The PrtS cysteine mutants were refolded and assessed functionally in reducing and oxidizing conditions and activity was assessed at pH 7.5 at 55°C. A, activity assays of the PrtS proteins refolded in the presence of DTT are shown and normalized to the activity observed with the wildtype protein. B, activity assays of the PrtS proteins refolded in the presence of a disulfide-enhancing additive are shown and normalized to the activity observed with the wildtype protein. C, the fold change between reducing and oxidizing conditions, A and B, is shown for the wildtype and mutant proteins. D, the PrtS protein were pre-incubated with maleimide and refolded as in C. Activity is shown normalized to the wildtype. Data shown are mean ± standard deviation from at least three independent experiments. Relative activity values were derived from the mean reaction velocities under steady state conditions with wildtype PrtS activity normalized to 1.0.
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pone.0138419.g005: Disulfide stabilization under relaxed biochemical conditions.The PrtS cysteine mutants were refolded and assessed functionally in reducing and oxidizing conditions and activity was assessed at pH 7.5 at 55°C. A, activity assays of the PrtS proteins refolded in the presence of DTT are shown and normalized to the activity observed with the wildtype protein. B, activity assays of the PrtS proteins refolded in the presence of a disulfide-enhancing additive are shown and normalized to the activity observed with the wildtype protein. C, the fold change between reducing and oxidizing conditions, A and B, is shown for the wildtype and mutant proteins. D, the PrtS protein were pre-incubated with maleimide and refolded as in C. Activity is shown normalized to the wildtype. Data shown are mean ± standard deviation from at least three independent experiments. Relative activity values were derived from the mean reaction velocities under steady state conditions with wildtype PrtS activity normalized to 1.0.

Mentions: The proteases were first refolded then diluted into reaction buffers at the indicated temperatures. Activity was first assessed under relaxed conditions where the wildtype protein showed high activity: pH 7.5 and 55°C. In the presence of DTT, small changes in PrtS activity could be seen using the fluorescent peptide substrates (Fig 5A). The mutants showed greater than 90% activity when compared to the wildtype, consistent with proper folding and activation from the DTT-reduced state. When refolded in the presence of a disulfide-promoting additive, the mutant protein showed a modest increase in protease activity when compared to the wildtype under identical conditions (Fig 5B). The A8C-V339C mutant showed a ~20% increase in protease activity while the L12C-R302C mutant showed a ~15% increase in protease activity, as compared to wildtype. The redox dependent changes in mutant protease activity were consistent with structural changes associated with putative disulfide formation by the paired cysteines and resulted in an increase of protease activity of approximately 20–25% (Fig 5C).


Interdomain Contacts and the Stability of Serralysin Protease from Serratia marcescens.

Zhang L, Morrison AJ, Thibodeau PH - PLoS ONE (2015)

Disulfide stabilization under relaxed biochemical conditions.The PrtS cysteine mutants were refolded and assessed functionally in reducing and oxidizing conditions and activity was assessed at pH 7.5 at 55°C. A, activity assays of the PrtS proteins refolded in the presence of DTT are shown and normalized to the activity observed with the wildtype protein. B, activity assays of the PrtS proteins refolded in the presence of a disulfide-enhancing additive are shown and normalized to the activity observed with the wildtype protein. C, the fold change between reducing and oxidizing conditions, A and B, is shown for the wildtype and mutant proteins. D, the PrtS protein were pre-incubated with maleimide and refolded as in C. Activity is shown normalized to the wildtype. Data shown are mean ± standard deviation from at least three independent experiments. Relative activity values were derived from the mean reaction velocities under steady state conditions with wildtype PrtS activity normalized to 1.0.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4574703&req=5

pone.0138419.g005: Disulfide stabilization under relaxed biochemical conditions.The PrtS cysteine mutants were refolded and assessed functionally in reducing and oxidizing conditions and activity was assessed at pH 7.5 at 55°C. A, activity assays of the PrtS proteins refolded in the presence of DTT are shown and normalized to the activity observed with the wildtype protein. B, activity assays of the PrtS proteins refolded in the presence of a disulfide-enhancing additive are shown and normalized to the activity observed with the wildtype protein. C, the fold change between reducing and oxidizing conditions, A and B, is shown for the wildtype and mutant proteins. D, the PrtS protein were pre-incubated with maleimide and refolded as in C. Activity is shown normalized to the wildtype. Data shown are mean ± standard deviation from at least three independent experiments. Relative activity values were derived from the mean reaction velocities under steady state conditions with wildtype PrtS activity normalized to 1.0.
Mentions: The proteases were first refolded then diluted into reaction buffers at the indicated temperatures. Activity was first assessed under relaxed conditions where the wildtype protein showed high activity: pH 7.5 and 55°C. In the presence of DTT, small changes in PrtS activity could be seen using the fluorescent peptide substrates (Fig 5A). The mutants showed greater than 90% activity when compared to the wildtype, consistent with proper folding and activation from the DTT-reduced state. When refolded in the presence of a disulfide-promoting additive, the mutant protein showed a modest increase in protease activity when compared to the wildtype under identical conditions (Fig 5B). The A8C-V339C mutant showed a ~20% increase in protease activity while the L12C-R302C mutant showed a ~15% increase in protease activity, as compared to wildtype. The redox dependent changes in mutant protease activity were consistent with structural changes associated with putative disulfide formation by the paired cysteines and resulted in an increase of protease activity of approximately 20–25% (Fig 5C).

Bottom Line: Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases.Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability.This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15219, United States of America.

ABSTRACT
The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX) family of toxins and virulence factors, which mediated virulence in E. coli, B. pertussis, and P. aeruginosa, as well as other animal and plant pathogens. The serralysin proteases are structurally dynamic and their folding is regulated by calcium binding to a C-terminal domain that defines the RTX family of proteins. Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases. Extending from this, stabilization of these interactions in the native structure may lead to hyperstabilization of the folded protein. To test this hypothesis, cysteine pairs were introduced into the N-terminal helix and the RTX domain and protease folding and activity were assessed. Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability. This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding. These data demonstrate that the thermal and chemical stability of these proteases is, in part, mediated by binding between the RTX domain and the N-terminal helix and demonstrate that stabilization of this interaction can further stabilize the active protease, leading to additional pH and thermal tolerance.

No MeSH data available.


Related in: MedlinePlus