Tulsidas Shenoy Rajesh, Thangamani Saravanan, Ho Bow, Sivaraman J, Ding Jeak Ling
Department of Biological Sciences, National University of Singapore, Singapore.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009 May 1;65(Pt 5):533-5. doi: 10.1107/S1744309109014420. Epub 2009 Apr 28.
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.
丝氨酸蛋白酶在宿主与病原体的相互作用中起主要作用。已知先天免疫系统以非特异性方式对入侵的病原体作出反应。丝氨酸蛋白酶级联反应是鲎先天免疫系统的重要组成部分。从圆尾鲎的肝胰腺中鉴定出丝氨酸蛋白酶抑制剂CrSPI同工型1（CrSPI-1），这是一种独特的非经典卡扎尔型抑制剂，分子量为9.3 kDa。它能有效抑制枯草杆菌蛋白酶，构成了针对入侵微生物的强大先天免疫防御。在此，报道了CrSPI-1与枯草杆菌蛋白酶的克隆、表达、纯化及共结晶。晶体衍射分辨率达到2.6 Å，属于空间群P2(1)，晶胞参数a = 73.8、b = 65.0、c = 111.9 Å，β = 95.4°。马修斯系数（V(M) = 2.64 ų Da⁻¹，对应溶剂含量53%）以及初步结构解析分析表明，不对称单元中存在一个异源三聚体（CrSPI-1与枯草杆菌蛋白酶的比例为1:2）和一个游离的枯草杆菌蛋白酶分子。
