Dial Ravina, Sun Zhen-Yu J, Freedman Steven J
Division of Hemostasis and Thrombosis and Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, Massachusetts 02215, USA.
Biochemistry. 2003 Aug 26;42(33):9937-45. doi: 10.1021/bi034989o.
Numerous transcription factors interact with the basal transcriptional machinery through the transcriptional co-activators p300 and CREB-binding protein (CBP). The Zn(2+)-binding cysteine/histidine-rich 1 (CH1) domain of p300/CBP binds many of these transcription factors, including hypoxia-inducible factor (HIF). We studied the structural and biophysical properties of the p300 CH1 domain alone and bound to the HIF-1 alpha C-terminal transactivation domain (TAD) to understand the diverse binding properties of CH1. The Zn(2+)-bound CH1 domain (CH1-Zn(2+)) and the HIF-1 alpha TAD-CH1 complex (CH1-Zn(2+)-HIF-1 alpha) are similarly helical, whereas metal-free CH1 is mostly random coil. CH1-Zn(2+) undergoes noncooperative thermal denaturation, does not have a near-UV elliptical signal, and binds the hydrophobic fluorophore ANS. In contrast, the CH1-Zn(2+)-HIF-1 alpha complex undergoes cooperative thermal denaturation, does produce a near-UV signal, and does not bind ANS. Addition of Zn(2+) ions to metal-free CH1 produced one conformational change, and subsequent addition of a HIF-1 alpha TAD peptide induced a second conformational change as detected by intrinsic tryptophan fluorescence spectroscopy. The NMR (1)H-(15)N HSQC spectrum of CH1-Zn(2+) exhibits few poorly dispersed peaks with broad line widths. Removal of metal ions produces more poorly dispersed peaks with sharper line widths. Addition of a HIF-1 alpha TAD peptide to CH1-Zn(2+) produces many well-dispersed peaks with sharp line widths. Taken together, these data support three conformational states for CH1, including an unstructured metal-free domain, a partially structured Zn(2+)-bound domain with molten globule characteristics, and a stable, well-ordered HIF-1 alpha TAD-CH1 complex.
众多转录因子通过转录共激活因子p300和CREB结合蛋白（CBP）与基础转录机制相互作用。p300/CBP的富含锌（2+）的半胱氨酸/组氨酸1（CH1）结构域结合许多此类转录因子，包括缺氧诱导因子（HIF）。我们研究了单独的p300 CH1结构域以及与HIF-1α C末端反式激活结构域（TAD）结合时的结构和生物物理特性，以了解CH1的多种结合特性。锌（2+）结合的CH1结构域（CH1-Zn(2+)）和HIF-1α TAD-CH1复合物（CH1-Zn(2+)-HIF-1α）具有相似的螺旋结构，而无金属的CH1大多为无规卷曲。CH1-Zn(2+)经历非协同热变性，没有近紫外椭圆信号，并且结合疏水荧光团ANS。相比之下，CH1-Zn(2+)-HIF-1α复合物经历协同热变性，确实产生近紫外信号，并且不结合ANS。向无金属的CH1中添加锌（2+）离子产生一种构象变化，随后添加HIF-1α TAD肽诱导第二种构象变化，这通过内在色氨酸荧光光谱检测到。CH1-Zn(2+)的核磁共振（1）H-（15）N HSQC谱显示很少有分散性差且线宽较宽的峰。去除金属离子会产生更多分散性差但线宽较窄的峰。向CH1-Zn(2+)中添加HIF-1α TAD肽会产生许多分散良好且线宽较窄的峰。综上所述，这些数据支持CH1的三种构象状态，包括无结构的无金属结构域、具有熔球特征的部分结构化的锌（2+）结合结构域以及稳定、有序的HIF-1α TAD-CH1复合物。
