Blair Rebecca H, Horn Abigail E, Pazhani Yogitha, Grado Lizbeth, Goodrich James A, Kugel Jennifer F
Department of Chemistry and Biochemistry, University of Colorado, 596 UCB, Boulder, CO 80309-0596, USA.
Department of Chemistry and Biochemistry, University of Colorado, 596 UCB, Boulder, CO 80309-0596, USA.
J Mol Biol. 2016 Oct 9;428(20):4060-4072. doi: 10.1016/j.jmb.2016.08.018. Epub 2016 Aug 21.
High mobility group box protein 1 (HMGB1) is an architectural protein that facilitates the formation of protein-DNA assemblies involved in transcription, recombination, DNA repair, and chromatin remodeling. Important to its function is the ability of HMGB1 to bend DNA non-sequence specifically. HMGB1 contains two HMG boxes that bind and bend DNA (the A box and the B box) and a C-terminal acidic tail. We investigated how these domains contribute to DNA bending by HMGB1 using single-molecule fluorescence resonance energy transfer (FRET), which enabled us to resolve heterogeneous populations of bent and unbent DNA. We found that full-length (FL) HMGB1 bent DNA more than the individual A and B boxes. Removing the C-terminal tail resulted in a protein that bent DNA to a greater extent than the FL protein. These data suggest that the A and B boxes simultaneously bind DNA in the absence of the C-terminal tail, but the tail modulates DNA binding and bending by one of the HMG boxes in the FL protein. Indeed, a construct composed of the B box and the C-terminal tail only bent DNA at higher protein concentrations. Moreover, in the context of the FL protein, mutating the A box such that it could not bend DNA resulted in a protein that bent DNA similar to a single HMG box and only at higher protein concentrations. We propose a model in which the HMGB1 C-terminal tail serves as an intramolecular damper that modulates the interaction of the B box with DNA.
高迁移率族蛋白B1（HMGB1）是一种结构蛋白，它促进参与转录、重组、DNA修复和染色质重塑的蛋白质-DNA组装体的形成。HMGB1非序列特异性弯曲DNA的能力对其功能很重要。HMGB1包含两个结合并弯曲DNA的HMG盒（A盒和B盒）以及一个C端酸性尾巴。我们使用单分子荧光共振能量转移（FRET）研究了这些结构域如何对HMGB1介导的DNA弯曲产生影响，这使我们能够解析弯曲和未弯曲DNA的异质群体。我们发现全长（FL）HMGB1比单个A盒和B盒更能弯曲DNA。去除C端尾巴后得到的一种蛋白质比FL蛋白质更能弯曲DNA。这些数据表明，在没有C端尾巴的情况下，A盒和B盒同时结合DNA，但尾巴调节FL蛋白质中一个HMG盒的DNA结合和弯曲。实际上，仅由B盒和C端尾巴组成的构建体仅在较高蛋白质浓度下才能弯曲DNA。此外，在FL蛋白质的背景下，将A盒突变使其不能弯曲DNA，会产生一种弯曲DNA的蛋白质，其弯曲程度类似于单个HMG盒，且仅在较高蛋白质浓度下才会出现。我们提出了一个模型，其中HMGB1的C端尾巴作为分子内阻尼器，调节B盒与DNA的相互作用。
