Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin.
Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulator...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2010-12-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3004852?pdf=render |
| _version_ | 1828767992275533824 |
|---|---|
| author | Pankaj Kumar Giri Xuhua Tang Saravanan Thangamani Rajesh T Shenoy Jeak Ling Ding Kunchithapadam Swaminathan J Sivaraman |
| author_facet | Pankaj Kumar Giri Xuhua Tang Saravanan Thangamani Rajesh T Shenoy Jeak Ling Ding Kunchithapadam Swaminathan J Sivaraman |
| author_sort | Pankaj Kumar Giri |
| collection | DOAJ |
| description | Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC(50) of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases. |
| first_indexed | 2024-12-11T07:38:44Z |
| format | Article |
| id | doaj.art-9d1dcbe97fdd49c688eca5b4e25e59ed |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-11T07:38:44Z |
| publishDate | 2010-12-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-9d1dcbe97fdd49c688eca5b4e25e59ed2022-12-22T01:15:39ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-12-01512e1525810.1371/journal.pone.0015258Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin.Pankaj Kumar GiriXuhua TangSaravanan ThangamaniRajesh T ShenoyJeak Ling DingKunchithapadam SwaminathanJ SivaramanProtease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC(50) of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases.http://europepmc.org/articles/PMC3004852?pdf=render |
| spellingShingle | Pankaj Kumar Giri Xuhua Tang Saravanan Thangamani Rajesh T Shenoy Jeak Ling Ding Kunchithapadam Swaminathan J Sivaraman Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin. PLoS ONE |
| title | Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin. |
| title_full | Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin. |
| title_fullStr | Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin. |
| title_full_unstemmed | Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin. |
| title_short | Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin. |
| title_sort | modifying the substrate specificity of carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin |
| url | http://europepmc.org/articles/PMC3004852?pdf=render |
| work_keys_str_mv | AT pankajkumargiri modifyingthesubstratespecificityofcarcinoscorpiusrotundicaudaserineproteaseinhibitordomain1totargetthrombin AT xuhuatang modifyingthesubstratespecificityofcarcinoscorpiusrotundicaudaserineproteaseinhibitordomain1totargetthrombin AT saravananthangamani modifyingthesubstratespecificityofcarcinoscorpiusrotundicaudaserineproteaseinhibitordomain1totargetthrombin AT rajeshtshenoy modifyingthesubstratespecificityofcarcinoscorpiusrotundicaudaserineproteaseinhibitordomain1totargetthrombin AT jeaklingding modifyingthesubstratespecificityofcarcinoscorpiusrotundicaudaserineproteaseinhibitordomain1totargetthrombin AT kunchithapadamswaminathan modifyingthesubstratespecificityofcarcinoscorpiusrotundicaudaserineproteaseinhibitordomain1totargetthrombin AT jsivaraman modifyingthesubstratespecificityofcarcinoscorpiusrotundicaudaserineproteaseinhibitordomain1totargetthrombin |