Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
PLoS One. 2013 Sep 5;8(9):e70562. doi: 10.1371/journal.pone.0070562. eCollection 2013.
In an effort to characterise the whole transcriptome of the fungus Hypocrea jecorina, cDNA clones of this fungus were identified that encode for previously unknown proteins that are likely to function in biomass degradation. One of these newly identified proteins, found to be co-regulated with the major H. jecorina cellulases, is a protein that was denoted Cellulose induced protein 1 (Cip1). This protein consists of a glycoside hydrolase family 1 carbohydrate binding module connected via a linker region to a domain with yet unknown function. After cloning and expression of Cip1 in H. jecorina, the protein was purified and biochemically characterised with the aim of determining a potential enzymatic activity for the novel protein. No hydrolytic activity against any of the tested plant cell wall components was found. The proteolytic core domain of Cip1 was then crystallised, and the three-dimensional structure of this was determined to 1.5 Å resolution utilising sulphur single-wavelength anomalous dispersion phasing (sulphor-SAD). A calcium ion binding site was identified in a sequence conserved region of Cip1 and is also seen in other proteins with the same general fold as Cip1, such as many carbohydrate binding modules. The presence of this ion was found to have a structural role. The Cip1 structure was analysed and a structural homology search was performed to identify structurally related proteins. The two published structures with highest overall structural similarity to Cip1 found were two poly-lyases: CsGL, a glucuronan lyase from H. jecorina and vAL-1, an alginate lyase from the Chlorella virus. This indicates that Cip1 may be a lyase. However, initial trials did not detect significant lyase activity for Cip1. Cip1 is the first structure to be solved of the 23 currently known Cip1 sequential homologs (with a sequence identity cut-off of 25%), including both bacterial and fungal members.
为了描绘真菌 Hypocrea jecorina 的整个转录组特征，鉴定了该真菌的 cDNA 克隆，这些克隆编码以前未知的、可能在生物质降解中起作用的蛋白质。在这些新鉴定的蛋白质中，有一种与主要的 Hypocrea jecorina 纤维素酶协同调节的蛋白质，被称为纤维素诱导蛋白 1（Cip1）。该蛋白由糖苷水解酶家族 1 碳水化合物结合模块组成，通过连接区与具有未知功能的结构域相连。在 Hypocrea jecorina 中克隆和表达 Cip1 后，对该蛋白进行了纯化和生化特性分析，目的是确定该新型蛋白的潜在酶活性。没有发现针对任何测试的植物细胞壁成分的水解活性。然后对 Cip1 的蛋白水解核心结构域进行了结晶，并利用硫单波长反常散射相位（硫-SAD）确定了该结构的三维结构，分辨率为 1.5 Å。在 Cip1 的序列保守区鉴定到一个钙离子结合位点，在具有与 Cip1 相同总体折叠的其他蛋白中也观察到该位点，如许多碳水化合物结合模块。发现该离子的存在具有结构作用。分析了 Cip1 结构，并进行了结构同源搜索以鉴定结构相关的蛋白。与 Cip1 具有最高整体结构相似性的两个已发表结构是两种聚裂解酶：来自 Hypocrea jecorina 的葡糖醛酸裂解酶 CsGL 和来自 Chlorella 病毒的藻酸盐裂解酶 vAL-1。这表明 Cip1 可能是一种裂解酶。然而，最初的试验未检测到 Cip1 的显著裂解酶活性。Cip1 是目前已知的 23 个 Cip1 序列同源物中第一个被解析的结构（序列同一性截止值为 25%），包括细菌和真菌成员。
