Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

Shenoy, Rajesh T.; Thangamani, Saravanan; Velazquez-Campoy, Adrian; Ho, Bow; Ding, Jeak Ling; Sivaraman, J.; Kursula, Petri

doi:10.1371/journal.pone.0018838

Title: Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

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Abstract

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki=1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease,more »« less

Authors:

Shenoy, Rajesh T. ^[1]; Thangamani, Saravanan ^[2]; Velazquez-Campoy, Adrian ^[3]; Ho, Bow ^[1]; Ding, Jeak Ling ^[1]; Sivaraman, J. ^[1]; Kursula, Petri ^[4]

National Univ. of Singapore (Singapore)
National Univ. of Singapore (Singapore); Univ. of Texas Medical Branch, Galveston, TX (United States)
Univ. of Zaragoza (Spain)
Univ. of Oulu (Germany)

Publication Date:: Tue Apr 26 00:00:00 EDT 2011

Research Org.:: Brookhaven National Lab. (BNL), Upton, NY (United States)

Sponsoring Org.:: USDOE Office of Science (SC)

OSTI Identifier:: 1069365

Report Number(s):: BNL-99937-2013-JA
Journal ID: ISSN 1932-6203

DOE Contract Number:: AC02-98CH10886

Resource Type:: Journal Article

Journal Name:: PLoS ONE

Additional Journal Information:: Journal Volume: 6; Journal Issue: 4; Journal ID: ISSN 1932-6203

Publisher:: Public Library of Science

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES

Citation Formats


                    Shenoy, Rajesh T., Thangamani, Saravanan, Velazquez-Campoy, Adrian, Ho, Bow, Ding, Jeak Ling, Sivaraman, J., and Kursula, Petri. Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin.  United States: N. p., 2011. 
        Web.  doi:10.1371/journal.pone.0018838.

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                    Shenoy, Rajesh T., Thangamani, Saravanan, Velazquez-Campoy, Adrian, Ho, Bow, Ding, Jeak Ling, Sivaraman, J., & Kursula, Petri. Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin.  United States.  https://doi.org/10.1371/journal.pone.0018838

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                    Shenoy, Rajesh T., Thangamani, Saravanan, Velazquez-Campoy, Adrian, Ho, Bow, Ding, Jeak Ling, Sivaraman, J., and Kursula, Petri. 2011.  
        "Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin".  United States.  https://doi.org/10.1371/journal.pone.0018838.

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@article{osti_1069365,

  title        = {Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin},

  author       = {Shenoy, Rajesh T. and Thangamani, Saravanan and Velazquez-Campoy, Adrian and Ho, Bow and Ding, Jeak Ling and Sivaraman, J. and Kursula, Petri},

  abstractNote = {Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki=1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1:2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.},

  doi          = {10.1371/journal.pone.0018838},

  url          = {https://www.osti.gov/biblio/1069365},
  journal      = {PLoS ONE},

  issn         = {1932-6203},

  number       = 4,

  volume       = 6,

  place        = {United States},

  year         = {Tue Apr 26 00:00:00 EDT 2011},

  month        = {Tue Apr 26 00:00:00 EDT 2011}

}

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