Eakin A E, McGrath M E, McKerrow J H, Fletterick R J, Craik C S
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
J Biol Chem. 1993 Mar 25;268(9):6115-8.
The major cysteine protease of Trypanosoma cruzi, cruzain, has been previously expressed in Escherichia coli as a fusion polypeptide. The proteolytic processing events required to obtain active, mature cruzain from the recombinant expression system have been characterized using mutational analysis of the cloned gene. An inactive variant of cruzain (cruzain-C25A) revealed that the proteolytic cleavage of the COOH-terminal domain from the recombinant cruzain is independent of cruzain activity. This cleavage event, presumably performed by another protease, was reduced, although not completely eliminated, in a variant in which the cleavage recognition site was altered (cruzain-E219P). To obtain a homogeneous COOH terminus of the recombinant enzyme, a truncated form of cruzain (cruzain-delta c) was engineered by insertion of a stop codon in the gene at a site corresponding to autoproteolysis observed with the native enzyme, purified from epimastigotes. Diffraction quality crystals of recombinant cruzain (cruzain) and the truncated variant (cruzain-delta c) have been produced and characterized. Cruzain and cruzain-delta c were cocrystallized with the peptide fluoromethyl ketone (FMK) inhibitors, Z-Phe-Arg-FMK and Z-Phe-Ala-FMK, respectively, (where Z is benzyloxycarbonyl). The crystals are monoclinic, space group P2(1), with a = 45.5 A, b = 51.0 A, c = 45.7 A, and beta = 116.1 degrees. One cruzain molecule is present in the asymmetric unit. The crystallographic data reveal that the high resolution structure determination is feasible. This system will facilitate the three-dimensional structure determinations and biochemical analyses of cruzain and cruzain variants.
克氏锥虫的主要半胱氨酸蛋白酶——克氏锥虫蛋白酶，此前已在大肠杆菌中作为融合多肽表达。利用克隆基因的突变分析，已对从重组表达系统中获得活性成熟克氏锥虫蛋白酶所需的蛋白水解加工事件进行了表征。克氏锥虫蛋白酶的一种无活性变体（克氏锥虫蛋白酶-C25A）表明，从重组克氏锥虫蛋白酶中切割COOH末端结构域与克氏锥虫蛋白酶活性无关。尽管在切割识别位点发生改变的变体（克氏锥虫蛋白酶-E219P）中，由另一种蛋白酶进行的这种切割事件有所减少，但并未完全消除。为了获得重组酶的均一COOH末端，通过在基因中对应于天然酶自蛋白水解的位点插入一个终止密码子，构建了一种截短形式的克氏锥虫蛋白酶（克氏锥虫蛋白酶-δc），该天然酶从无鞭毛体中纯化得到。已制备并表征了重组克氏锥虫蛋白酶（克氏锥虫蛋白酶）和截短变体（克氏锥虫蛋白酶-δc）的衍射质量晶体。克氏锥虫蛋白酶和克氏锥虫蛋白酶-δc分别与肽氟甲基酮（FMK）抑制剂Z-Phe-Arg-FMK和Z-Phe-Ala-FMK共结晶（其中Z为苄氧羰基）。晶体为单斜晶系，空间群P2(1)，a = 45.5 Å，b = 51.0 Å，c = 45.7 Å，β = 116.1°。不对称单元中存在一个克氏锥虫蛋白酶分子。晶体学数据表明，高分辨率结构测定是可行的。该系统将有助于克氏锥虫蛋白酶和克氏锥虫蛋白酶变体的三维结构测定和生化分析。
