Matthews J R, Kaszubska W, Turcatti G, Wells T N, Hay R T
School of Biological and Medical Sciences, University of St Andrews, Fife, UK.
Nucleic Acids Res. 1993 Apr 25;21(8):1727-34. doi: 10.1093/nar/21.8.1727.
A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunit of NF-kappa B. Differential labelling with [14C] iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition site of the protein. To determine the importance of this cysteine residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined. Scatchard analysis indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an intermediate affinity. Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly. Competition analyses with oligonucleotide variants of the DNA recognition site and nonspecific E. coli DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was impaired in its ability to discriminate between specific and non-specific DNA. Thus the sulphydryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappa B.
已开发出一种强大的化学修饰程序来确定NF-κB p50亚基对DNA识别的决定因素。用[14C]碘乙酸进行差异标记已鉴定出一个保守的半胱氨酸残基Cys62，该残基因存在含有该蛋白质特异性识别位点的寡核苷酸而免受修饰。为了确定这个半胱氨酸残基的重要性，将p50中每个保守的半胱氨酸都替换为丝氨酸，并测定突变蛋白的DNA结合特性。Scatchard分析表明，C62S突变体与其DNA识别位点结合的解离常数比野生型蛋白大10倍，而其他两个突变体以中等亲和力结合。解离速率常数测量结果与野生型、C119S和C273S p50蛋白的解离常数相关性良好，而p50 C62S-DNA复合物异常快速地解离。用DNA识别位点的寡核苷酸变体和非特异性大肠杆菌DNA进行的竞争分析表明，C62S p50突变体的DNA结合位点特异性发生了改变，并且其区分特异性和非特异性DNA的能力受损。因此，Cys62的巯基是NF-κB p50亚基对DNA识别的重要决定因素。
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