Hua Q X, Jia W H, Bullock B P, Habener J F, Weiss M A
Department of Biochemistry, Center for Molecular Oncology, The University of Chicago, Illinois 60637, USA.
Biochemistry. 1998 Apr 28;37(17):5858-66. doi: 10.1021/bi9800808.
A model of transcriptional activator-coactivator recognition is provided by the mammalian CREB activation domain and the KIX domain of coactivator CBP. The CREB kinase-inducible activation domain (pKID, 60 residues) is disordered in solution and undergoes an alpha-helical folding transition on binding to CBP [Radhakrishan, I., Perez-Alvarado, G. C., Parker, D., Dyson, H. J., Montminy, M. R., and Wright, P. E. (1997) Cell 91, 741-752]. Binding requires phosphorylation of a conserved serine (RPpSYR) in pKID associated in vivo with the biological activation of CREB signaling pathways. The CBP-bound structure of CREB contains two alpha-helices (designated alphaA and alphaB) flanking the phosphoserine; the bound structure is stabilized by specific interactions with CBP. Here, the nascent structure of an unbound pKID domain is characterized by multidimensional NMR spectroscopy. The solubility of the phosphopeptide (46 residues) was enhanced by truncation of N- and C-terminal residues not involved in pKID-CBP interactions. Although disordered under physiologic conditions, the pKID fragment and its unphosphorylated parent peptide exhibit partial folding at low temperatures. One recognition helix (alphaA) is well-defined at 4 degreesC, whereas the other (alphaB) is disordered but inducible in 40% trifluoroethanol (TFE). Such nascent structure is independent of serine phosphorylation and correlates with the relative extent of engagement of the two alpha-helices in the pKID-KIX complex; whereas alphaA occupies a peripheral binding site with few intermolecular contacts, the TFE-inducible alphaB motif is deeply engaged in a hydrophobic groove. Our results support the use of TFE as an empirical probe of hidden structural propensities and define a correspondence between induced fit and the nascent structure of peptide fragments.
哺乳动物CREB激活结构域和辅激活因子CBP的KIX结构域提供了一种转录激活因子-辅激活因子识别模型。CREB激酶诱导激活结构域（pKID，60个氨基酸残基）在溶液中无序，与CBP结合时发生α-螺旋折叠转变[Radhakrishan, I., Perez-Alvarado, G. C., Parker, D., Dyson, H. J., Montminy, M. R., and Wright, P. E. (1997) Cell 91, 741 - 752]。结合需要pKID中一个保守丝氨酸（RPpSYR）的磷酸化，该丝氨酸在体内与CREB信号通路的生物学激活相关。与CBP结合的CREB结构包含两个位于磷酸丝氨酸两侧的α-螺旋（分别命名为αA和αB）；结合结构通过与CBP的特异性相互作用得以稳定。在此，通过多维核磁共振光谱对未结合的pKID结构域的新生结构进行了表征。通过截去不参与pKID-CBP相互作用的N端和C端残基，提高了磷酸肽（46个氨基酸残基）的溶解度。尽管在生理条件下无序，但pKID片段及其未磷酸化的母体肽在低温下表现出部分折叠。一个识别螺旋（αA）在4℃时结构明确，而另一个（αB）无序，但在40%三氟乙醇（TFE）中可诱导形成结构。这种新生结构与丝氨酸磷酸化无关，并且与pKID-KIX复合物中两个α-螺旋的相对结合程度相关；αA占据一个几乎没有分子间接触的外周结合位点，而TFE诱导的αB基序则深深地嵌入一个疏水凹槽中。我们的结果支持将TFE用作隐藏结构倾向的经验性探针，并确定了诱导契合与肽片段新生结构之间的对应关系。
