Wikström A, Berglund H, Hambraeus C, van den Berg S, Härd T
Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
J Mol Biol. 1999 Jun 18;289(4):963-79. doi: 10.1006/jmbi.1999.2806.
We examined the internal mobility of the estrogen receptor DNA-binding domain (ER DBD) using NMR15N relaxation measurements and compared it to that of the glucocorticoid receptor DNA-binding domain (GR DBD). The studied protein fragments consist of residues Arg183-His267 of the human ER and residues Lys438-Gln520 of the rat GR. The15N longitudinal (R1) and transverse (R2) relaxation rates and steady state {1H}-15N nuclear Overhauser enhancements (NOEs) were measured at 30 degrees C at1H NMR frequencies of 500 and 600 MHz. The NOE versus sequence profile and calculated order parameters for ER DBD backbone motions indicate enhanced internal dynamics on pico- to nanosecond time-scales in two regions of the core DBD. These are the extended strand which links the DNA recognition helix to the second zinc domain and the larger loop region of the second zinc domain. The mobility of the corresponding regions of the GR DBD, in particular that of the second zinc domain, is more limited. In addition, we find large differences between the ER and GR DBDs in the extent of conformational exchange mobility on micro- to millisecond time-scales. Based on measurements of R2as a function of the15N refocusing (CPMG) delay and quantitative (Lipari-Szabo-type) analysis, we conclude that conformational exchange occurs in the loop of the first zinc domain and throughout most of the second zinc domain of the ER DBD. The conformational exchange dynamics in GR DBD is less extensive and localized to two sites in the second zinc domain. The different dynamical features seen in the two proteins is consistent with previous studies of the free state structures in which the second zinc domain in the ER DBD was concluded to be disordered whereas the corresponding region of the GR DBD adopts a stable fold. Moreover, the regions of the ER DBD that undergo conformational dynamics on the micro- to millisecond time-scales in the free state are involved in intermolecular protein-DNA and protein-protein interactions in the dimeric bound state. Based on the present data and the previously published dynamical and DNA binding properties of a GR DBD triple mutant which recognize an ER binding site on DNA, we argue that the free state dynamical properties of the nuclear receptor DBDs is an important element in molecular recognition upon DNA binding.
我们使用核磁共振15N弛豫测量技术研究了雌激素受体DNA结合结构域（ER DBD）的内部流动性，并将其与糖皮质激素受体DNA结合结构域（GR DBD）的流动性进行了比较。所研究的蛋白质片段包括人ER的第183位精氨酸至第267位组氨酸残基以及大鼠GR的第438位赖氨酸至第520位谷氨酰胺残基。在30℃、1H NMR频率为500和600 MHz的条件下测量了15N纵向（R1）和横向（R2）弛豫率以及稳态{1H}-15N核Overhauser效应（NOE）。ER DBD主链运动的NOE与序列图谱及计算得到的序参数表明，在核心DBD的两个区域中，皮秒至纳秒时间尺度上的内部动力学增强。这两个区域分别是连接DNA识别螺旋与第二个锌结构域的延伸链以及第二个锌结构域的较大环区。GR DBD相应区域的流动性，尤其是第二个锌结构域的流动性，更为受限。此外，我们发现ER和GR DBD在微秒至毫秒时间尺度上的构象交换流动性程度存在很大差异。基于R2作为15N重聚焦（CPMG）延迟的函数的测量以及定量（Lipari-Szabo型）分析，我们得出结论，ER DBD的第一个锌结构域环区以及第二个锌结构域的大部分区域都发生了构象交换。GR DBD中的构象交换动力学范围较小，局限于第二个锌结构域的两个位点。这两种蛋白质中观察到的不同动力学特征与之前对自由态结构的研究一致，在之前的研究中得出结论，ER DBD中的第二个锌结构域是无序的，而GR DBD的相应区域则呈现出稳定的折叠结构。此外，在自由态下经历微秒至毫秒时间尺度构象动力学的ER DBD区域在二聚体结合态中参与分子间蛋白质-DNA和蛋白质-蛋白质相互作用。基于目前的数据以及先前发表的识别DNA上ER结合位点的GR DBD三重突变体的动力学和DNA结合特性，我们认为核受体DBD的自由态动力学特性是DNA结合时分子识别的一个重要因素。
