S1 pocket of a bacterially derived subtilisin-like protease underpins effective tissue destruction

Wilson Wong, Lakshmi C Wijeyewickrema, Ruth M Kennan, Shane B Reeve, David L Steer, Cyril Reboul, Alexander Ian Smith, Robert N Pike, Julian I Rood, James C Whisstock, Corrine J Porter

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96 sequence identity. Currently, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high-resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic, but smaller than that of BprB. We show that BprV is more effective in degrading extracellular matrix components of the host tissue than BprB. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1-Leu containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process.
Original languageEnglish
Pages (from-to)42180 - 42187
Number of pages8
JournalJournal of Biological Chemistry
Volume286
Issue number49
DOIs
Publication statusPublished - 2011

Cite this

Wong, W., Wijeyewickrema, L. C., Kennan, R. M., Reeve, S. B., Steer, D. L., Reboul, C., ... Porter, C. J. (2011). S1 pocket of a bacterially derived subtilisin-like protease underpins effective tissue destruction. Journal of Biological Chemistry, 286(49), 42180 - 42187. https://doi.org/10.1074/jbc.M111.298711
Wong, Wilson ; Wijeyewickrema, Lakshmi C ; Kennan, Ruth M ; Reeve, Shane B ; Steer, David L ; Reboul, Cyril ; Smith, Alexander Ian ; Pike, Robert N ; Rood, Julian I ; Whisstock, James C ; Porter, Corrine J. / S1 pocket of a bacterially derived subtilisin-like protease underpins effective tissue destruction. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 49. pp. 42180 - 42187.
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title = "S1 pocket of a bacterially derived subtilisin-like protease underpins effective tissue destruction",
abstract = "The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96 sequence identity. Currently, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high-resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic, but smaller than that of BprB. We show that BprV is more effective in degrading extracellular matrix components of the host tissue than BprB. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1-Leu containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process.",
author = "Wilson Wong and Wijeyewickrema, {Lakshmi C} and Kennan, {Ruth M} and Reeve, {Shane B} and Steer, {David L} and Cyril Reboul and Smith, {Alexander Ian} and Pike, {Robert N} and Rood, {Julian I} and Whisstock, {James C} and Porter, {Corrine J}",
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S1 pocket of a bacterially derived subtilisin-like protease underpins effective tissue destruction. / Wong, Wilson; Wijeyewickrema, Lakshmi C; Kennan, Ruth M; Reeve, Shane B; Steer, David L; Reboul, Cyril; Smith, Alexander Ian; Pike, Robert N; Rood, Julian I; Whisstock, James C; Porter, Corrine J.

In: Journal of Biological Chemistry, Vol. 286, No. 49, 2011, p. 42180 - 42187.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - S1 pocket of a bacterially derived subtilisin-like protease underpins effective tissue destruction

AU - Wong, Wilson

AU - Wijeyewickrema, Lakshmi C

AU - Kennan, Ruth M

AU - Reeve, Shane B

AU - Steer, David L

AU - Reboul, Cyril

AU - Smith, Alexander Ian

AU - Pike, Robert N

AU - Rood, Julian I

AU - Whisstock, James C

AU - Porter, Corrine J

PY - 2011

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N2 - The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96 sequence identity. Currently, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high-resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic, but smaller than that of BprB. We show that BprV is more effective in degrading extracellular matrix components of the host tissue than BprB. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1-Leu containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process.

AB - The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96 sequence identity. Currently, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high-resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic, but smaller than that of BprB. We show that BprV is more effective in degrading extracellular matrix components of the host tissue than BprB. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1-Leu containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process.

UR - http://www.jbc.org/content/286/49/42180.full.pdf+html

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DO - 10.1074/jbc.M111.298711

M3 - Article

VL - 286

SP - 42180

EP - 42187

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 1083-351X

IS - 49

ER -