A biophysical characterisation of factors controlling dimerisation and selectivity in the NF-κB and NFAT families
A biophysical characterisation of factors controlling dimerisation and selectivity in the NF-κB and NFAT families
复制标题
DOI：
10.1016/j.jmb.2004.03.083
复制
发表时间：
2004-06-18
影响因子：
5.6
通讯作者：
Blackburn, JM
中科院分区：
文献类型：
--
作者：
de Lumley, M;Hart, DJ;Blackburn, JM
真核转录因子Rel/NF-kappaB家族以高特异性和亲和力结合DNA作为同源或异源二聚体，介导多种生物过程。相比之下，活化t细胞核因子（NFAT）家族由于DNA结合结构域之间的结构相似性而被认为是Rel同源物，但它们以低亲和力单体结合DNA。NF-kappaB和NFAT家族之间的结构和功能重叠表明，它们可能从一个共同的单体祖先进化而来，但进化出不同的机制来实现与目标DNA序列的高亲和力结合。为了了解这些机制差异的起源，我们基于分子模型构建了两种嵌合蛋白，包括NFAT的dna结合结构域和NF-kappaB p50的二聚化结构域，仅在剪接位点的位置不同。野生型和嵌合蛋白的生物物理特性表明，其中一个嵌合体结合DNA为高亲和力的nf - kappab样合作二聚体，而另一个嵌合体结合为低亲和力的nfat样单体，这表明结构域间连接体在控制NFATc形成二聚体的内在能力方面的重要性。此外，我们还研究了预成型NF-kappaB二聚体之间的单体交换速率，并首次确定了NF-kappaB p50的本征二聚常数。这些数据支持一种模型，即NF-kappaB蛋白在体外和体内以高亲和力的预制同源或异源二聚体结合DNA，在未结合的形式下，它们仍然可以在体内生理相关的时间尺度上交换单体单位。(C) 2004 Elsevier Ltd.版权所有。
The Rel/NF-kappaB family of eukaryotic transcription factors bind DNA with high specificity and affinity as homo- or heterodimers to mediate a diverse range of biological processes. By comparison, the nuclear factor of activated T-cells (NFAT) family has been recognised as Rel homologues due to structural similarities between the DNA-binding domains, yet they bind DNA as lower-affinity monomers. The structural and functional overlap between the NF-kappaB and NFAT families suggests that they may be evolutionarily divergent from a common, monomeric ancestor but have evolved different mechanisms to achieve high-affinity binding to their target DNA sequences. In order to understand the origin of these mechanistic differences, we constructed two chimeric proteins, based on molecular modelling, comprising the DNA-binding domain of NFAT and the dimerisation domain of NF-kappaB p50, differing only in the position of the splice site. Biophysical characterisation of the wild-type and chimeric proteins revealed that one of the chimeras bound DNA as a high-affinity, NF-kappaB-like cooperative dimer, whilst the other bound as a lower-affinity, NFAT-like monomer, demonstrating the importance of the interdomain linker in controlling the intrinsic ability of NFATc to form dimers. In addition, we have studied the rate of exchange of monomers between preformed NF-kappaB dimers and have determined, for the first time, the intrinsic homodimerisation constant for NF-kappaB p50. These data support a model in which NF-kappaB proteins bind DNA both in vitro and in vivo as high-affinity preformed homo- or heterodimers, which in an unbound form can still exchange monomer units on a physiologically relevant timescale in vivo. (C) 2004 Elsevier Ltd. All rights reserved.