De Soumya, 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, BC V6T 1Z3, Canada.
Department of Oncological Sciences, School of Medicine, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112-5550, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815-6789, USA.
J Mol Biol. 2016 Apr 24;428(8):1515-30. doi: 10.1016/j.jmb.2016.02.020. Epub 2016 Feb 23.
The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge-dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity.
ETS转录抑制因子ETV6（或TEL）被一个α-螺旋自抑制，该α-螺旋在空间上阻碍其DNA结合ETS结构域。抑制性螺旋稳定性较差，当ETV6与特异性或非特异性DNA结合时会展开。利用核磁共振光谱，我们发现抑制性螺旋的折叠需要与ETS结构域的H1螺旋形成一个埋藏的电荷-偶极相互作用。这种相互作用还通过排除结合DNA的磷酸二酯主链与H1螺旋N端之间高度保守的偶极增强氢键，直接促成自抑制。为了进一步探究自抑制的热力学基础，我们生成了ETV6变体，在抑制性螺旋暴露于溶剂的表面引入了氨基酸替换。这些变化旨在增加抑制性螺旋的固有螺旋倾向，同时不干扰其与ETS结构域的堆积相互作用。核磁共振监测的酰胺氢交换测量结果证实，折叠后的抑制性螺旋的稳定性随着添加促进螺旋的替换而逐渐增加。这也导致自抑制逐渐增强，DNA结合亲和力降低。令人惊讶的是，通过二硫键将抑制性螺旋锁定在ETS结构域上会严重损害，但不会消除DNA结合。仍会通过一个偏离经典ETS结构域DNA结合表面的界面发生弱相互作用。总的来说，这些研究建立了抑制性螺旋稳定性与ETV6自抑制之间的直接热力学联系，并证明螺旋展开并不严格先于DNA结合。调节抑制性螺旋的稳定性为体内调节ETV6活性提供了一条潜在途径。
