title: Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin.
abstract: 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.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: Pankaj Kumar
authors.initials: PK
authors.lastname: Giri
authors.forename: Xuhua
authors.initials: X
authors.lastname: Tang
authors.forename: Saravanan
authors.initials: S
authors.lastname: Thangamani
authors.forename: Rajesh T
authors.initials: RT
authors.lastname: Shenoy
authors.forename: Jeak Ling
authors.initials: JL
authors.lastname: Ding
authors.forename: Kunchithapadam
authors.initials: K
authors.lastname: Swaminathan
authors.forename: J
authors.initials: J
authors.lastname: Sivaraman
mesh_terms: D000818:Animals; D002151:Calorimetry / Q000379:methods; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D006737:Horseshoe Crabs / Q000201:enzymology*; D006860:Hydrogen Bonding; D020128:Inhibitory Concentration 50; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
chemical_list: D015842:Serine Proteinase Inhibitors; D012694:Serine; D014357:Trypsin; D013917:Thrombin