Xhani Suela, Esaki Shingo, Huang Kenneth, Erlitzki Noa, Poon Gregory M K
Department of Chemistry, Georgia State University , Atlanta, Georgia 30303, United States.
Center for Diagnostics and Therapeutics, Georgia State University , Atlanta, Georgia 30303, United States.
J Phys Chem B. 2017 Apr 6;121(13):2748-2758. doi: 10.1021/acs.jpcb.7b00325. Epub 2017 Mar 28.
The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. Unlike other ETS homologues, such as Ets-1, DNA recognition by PU.1 is highly sensitive to its osmotic environment due to excess interfacial hydration in the complex. To investigate interfacial hydration in the two homologues, we mutated a conserved tyrosine residue, which is exclusively engaged in coordinating a well-defined water contact between the protein and DNA among ETS proteins, to phenylalanine. The loss of this water-mediated contact blunted the osmotic sensitivity of PU.1/DNA binding, but did not alter binding under normo-osmotic conditions, suggesting that PU.1 has evolved to maximize osmotic sensitivity. The homologous mutation in Ets-1, which was minimally sensitive to osmotic stress due to a sparsely hydrated interface, reduced DNA-binding affinity at normal osmolality but the complex became stabilized by osmotic stress. Molecular dynamics simulations of wildtype and mutant PU.1 and Ets-1 in their free and DNA-bound states, which recapitulated experimental features of the proteins, showed that abrogation of this tyrosine-mediated water contact perturbed the Ets-1/DNA complex not through disruption of interfacial hydration, but by inhibiting local dynamics induced specifically in the bound state. Thus, a configurationally identical water-mediated contact plays mechanistically distinct roles in mediating DNA recognition by structurally homologous ETS transcription factors.
ETS转录因子家族是一组功能异质性的基因调节因子，它们共享一个结构保守的、以其命名的DNA结合结构域。与其他ETS同源物（如Ets-1）不同，由于复合物中存在过量的界面水合作用，PU.1对DNA的识别对其渗透环境高度敏感。为了研究这两种同源物中的界面水合作用，我们将一个保守的酪氨酸残基突变为苯丙氨酸，该酪氨酸残基专门参与协调ETS蛋白中蛋白质与DNA之间明确的水接触。这种水介导接触的丧失减弱了PU.1/DNA结合的渗透敏感性，但在等渗条件下并未改变结合，这表明PU.1已经进化到最大限度地提高渗透敏感性。Ets-1中的同源突变由于界面水合作用稀少而对渗透应激敏感性最低，在正常渗透压下降低了DNA结合亲和力，但复合物在渗透应激下变得稳定。野生型和突变型PU.1以及Ets-1在游离和与DNA结合状态下的分子动力学模拟重现了蛋白质的实验特征，结果表明，这种酪氨酸介导的水接触的消除不是通过破坏界面水合作用，而是通过抑制在结合状态下特异性诱导的局部动力学来干扰Ets-1/DNA复合物。因此，在介导结构同源的ETS转录因子对DNA的识别过程中，构型相同的水介导接触发挥着机制上不同的作用。
