Open AccessCrystallization of a nonclassical Kazal‐type Carcinoscorpius rotundicauda serine protease inhibitor, CrSPI‐1, complexed with subtilisin
Author(s) -
Tulsidas Shenoy Rajesh,
Thangamani Saravanan,
Ho Bow,
Sivaraman J.,
Ding Jeak Ling
Publication year - 2009
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309109014420
Subject(s) - subtilisin , serine protease , proteases , serine , biology , innate immune system , protease , horseshoe crab , biochemistry , chemistry , enzyme , receptor , ecology
Serine proteases play a major role in host–pathogen interactions. The innate immune system is known to respond to invading pathogens in a nonspecific manner. The serine protease cascade is an essential component of the innate immune system of the horseshoe crab. The serine protease inhibitor CrSPI isoform 1 (CrSPI‐1), a unique nonclassical Kazal‐type inhibitor of molecular weight 9.3 kDa, was identified from the hepatopancreas of the horseshoe crab Carcinoscorpius rotundicauda . It potently inhibits subtilisin and constitutes a powerful innate immune defence against invading microbes. Here, the cloning, expression, purification and cocrystallization of CrSPI‐1 with subtilisin are reported. The crystals diffracted to 2.6 Å resolution and belonged to space group P 2 1 , with unit‐cell parameters a = 73.8, b = 65.0, c = 111.9 Å, β = 95.4°. The Matthews coefficient ( V M = 2.64 Å 3 Da −1 , corresponding to 53% solvent content) and analysis of the preliminary structure solution indicated the presence of one heterotrimer (1:2 ratio of CrSPI‐1:subtilisin) and one free subtilisin molecule in the asymmetric unit.