Friedler Assaf, Veprintsev Dmitry B, Freund Stefan M V, von Glos Karoly I, Fersht Alan R
MRC Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, United Kingdom.
Structure. 2005 Apr;13(4):629-36. doi: 10.1016/j.str.2005.01.020.
The binding of nonspecific DNA to the C-terminal negative regulatory domain (CTD) of p53 modulates its activity. The CTD is a natively unfolded region, which is subject to acetylation and phosphorylation at several residues as part of control. To measure the effect of covalent modification on binding to DNA, we synthesized a series of fluorescein-labeled CTD peptides with single and multiple acetylations at lysine residues that we had identified by NMR as making contact with DNA, and developed an analytical ultracentrifugation method to study their binding to DNA. Binding depended on ionic strength, indicating an electrostatic contribution. Monoacetylation weakened DNA binding at physiological ionic strength 2- to 3-fold, diacetylations resulted in further 2- to 3-fold decrease in the affinity, and tri- and tetraacetylations rendered DNA binding undetectable. Phosphorylation at S392 did not affect DNA binding. NMR spectroscopy showed binding to DNA did not induce significant structure into CTD, apart possibly from local helix formation.
非特异性DNA与p53的C端负调控结构域（CTD）的结合会调节其活性。CTD是一个天然未折叠区域，作为调控的一部分，该区域的几个残基会发生乙酰化和磷酸化。为了测量共价修饰对与DNA结合的影响，我们合成了一系列在赖氨酸残基处有单个和多个乙酰化修饰的荧光素标记的CTD肽，这些赖氨酸残基已通过核磁共振（NMR）鉴定为与DNA接触的位点，并且开发了一种分析超速离心方法来研究它们与DNA的结合。结合作用取决于离子强度，表明存在静电作用。单乙酰化在生理离子强度下使DNA结合减弱2至3倍，双乙酰化导致亲和力进一步降低2至3倍，而三乙酰化和四乙酰化则使DNA结合无法检测到。S392位点的磷酸化不影响DNA结合。核磁共振光谱显示，与DNA的结合除了可能导致局部螺旋形成外，不会诱导CTD形成显著的结构。
