Hu D, Crist M, Duan X, Quiocho F A, Gimble F S
Center for Genome Research, Institute of Biosciences and Technology, Department of Medical Biochemistry, The Texas A & M University System Health Science Center, Houston, Texas 77030, USA.
J Biol Chem. 2000 Jan 28;275(4):2705-12. doi: 10.1074/jbc.275.4.2705.
The PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure. To determine the spatial relationship between bases in the PI-SceI recognition sequence and selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by photocross-linking and affinity cleavage methods. Unique solvent-accessible cysteine residues were introduced into the two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid. The results suggest that an extended beta-hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate. To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein that bind DNA. The data strongly support a model of the PI-SceI-DNA complex derived from this structure.
PI-SceI蛋白是一种内含肽编码的归巢内切核酸酶，它通过在酵母基因组中的单个位点产生双链断裂来启动其基因的移动性。PI-SceI蛋白的剪接和内切核酸酶活性位点分别位于PI-SceI结构的两个结构域中。为了确定PI-SceI识别序列中的碱基与选定的PI-SceI氨基酸之间的空间关系，采用光交联和亲和切割方法对PI-SceI-DNA复合物进行了探测。在PI-SceI的两个结构域中的第91、97、170、230、376和378位引入了独特的可溶剂接触的半胱氨酸残基，并用4-叠氮苯甲酰溴或铁（S）-1-（对溴乙酰氨基苄基）-乙二胺四乙酸（FeBABE）对突变蛋白进行了修饰。与苯叠氮偶联的蛋白与PI-SceI靶序列发生交联，而FeBABE修饰的蛋白切割衍生氨基酸附近的DNA。结果表明，内切核酸酶结构域中包含376和378位残基的延伸β-发夹环与PI-SceI切割位点附近的大沟接触。相反，蛋白剪接结构域中的91、97和170位残基与底物的一个远距离区域紧密相邻。为了解释我们的结果，我们使用了一种新的PI-SceI结构，该结构在与DNA结合的蛋白区域中是有序的。这些数据有力地支持了从该结构推导出来的PI-SceI-DNA复合物模型。
