Desjardins Geneviève, Okon Mark, Graves Barbara J, McIntosh Lawrence P
Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia , Vancouver, British Columbia V6T 1Z3, Canada.
Department of Oncological Sciences, University of Utah School of Medicine, Huntsman Cancer Institute, University of Utah , Salt Lake City, Utah 84112-5550, United States.
Biochemistry. 2016 Jul 26;55(29):4105-18. doi: 10.1021/acs.biochem.6b00460. Epub 2016 Jul 15.
The affinity of the Ets-1 transcription factor for DNA is autoinhibited by an intrinsically disordered serine-rich region (SRR) and a helical inhibitory module (IM) appended to its winged helix-turn-helix ETS domain. Using NMR spectroscopy, we investigated how Ets-1 recognizes specific versus nonspecific DNA, with a focus on the roles of protein dynamics and autoinhibition in these processes. Upon binding either DNA, the two marginally stable N-terminal helices of the IM predominantly unfold, but still sample partially ordered conformations. Also, on the basis of amide chemical shift perturbation mapping, Ets-1 associates with both specific and nonspecific DNA through the same canonical ETS domain interface. These interactions are structurally independent of the SRR, and thus autoinhibition does not impart DNA-binding specificity. However, relative to the pronounced NMR spectroscopic changes in Ets-1 resulting from specific DNA binding, the spectra of the nonspecific DNA complexes showed conformational exchange broadening and lacked several diagnostic amide and indole signals attributable to hydrogen bonding interactions seen in reported X-ray crystallographic structures of this transcription factor with its cognate DNA sequences. Such differences are highlighted by the chemical shift and relaxation properties of several interfacial lysine and arginine side chains. Collectively, these data support a general model in which Ets-1 interacts with nonspecific DNA via dynamic electrostatic interactions, whereas hydrogen bonding drives the formation of well-ordered complexes with specific DNA.
Ets-1转录因子对DNA的亲和力受到一个内在无序的富含丝氨酸区域（SRR）和一个附加在其带翼螺旋-转角-螺旋ETS结构域上的螺旋抑制模块（IM）的自动抑制。我们使用核磁共振光谱研究了Ets-1如何识别特异性和非特异性DNA，重点关注蛋白质动力学和自动抑制在这些过程中的作用。在结合任何一种DNA时，IM的两个稳定性稍差的N端螺旋主要展开，但仍部分处于有序构象。此外，基于酰胺化学位移扰动图谱，Ets-1通过相同的经典ETS结构域界面与特异性和非特异性DNA结合。这些相互作用在结构上独立于SRR，因此自动抑制并不赋予DNA结合特异性。然而，相对于特异性DNA结合导致的Ets-1明显的核磁共振光谱变化，非特异性DNA复合物的光谱显示出构象交换加宽，并且缺乏一些可归因于该转录因子与其同源DNA序列的报道X射线晶体结构中所见氢键相互作用的诊断性酰胺和吲哚信号。几个界面赖氨酸和精氨酸侧链的化学位移和弛豫特性突出了这些差异。总体而言，这些数据支持一个通用模型，即Ets-1通过动态静电相互作用与非特异性DNA相互作用，而氢键驱动与特异性DNA形成有序复合物。
