Crystallization of a nonclassical Kazal-type Carcinoscorpius rotundicauda serine protease inhibitor, CrSPI-1, complexed with subtilisin.
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 A resolution and belonged to space group P2(1), with unit-cell parameters a = 73.8, b = 65.0, c = 111.9 A, beta = 95.4 degrees . The Matthews coefficient (V(M) = 2.64 A(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.
Tulsidas
Shenoy Rajesh
SR
Department of Biological Sciences, National University of Singapore, Singapore.
Thangamani
Saravanan
S
Ho
Bow
B
Sivaraman
J
J
Ding
Jeak Ling
JL
D000818:Animals; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D006737:Horseshoe Crabs / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020860:Subtilisin / Q000737:chemistry* / Q000378:metabolism*
D015842:Serine Proteinase Inhibitors; D020860:Subtilisin