Division of Plant Biology, Bose Institute, Kolkata, 700054, India.
Department of Chemistry, Bose Institute, Kolkata, 700054, India.
Plant Mol Biol. 2016 Oct;92(3):371-88. doi: 10.1007/s11103-016-0519-y. Epub 2016 Aug 9.
ARID-HMG DNA-binding proteins represent a novel group of HMG-box containing protein family where the AT-rich interaction domain (ARID) is fused with the HMG-box domain in a single polypeptide chain. ARID-HMG proteins are highly plant specific with homologs found both in flowering plants as well as in moss such as Physcomitrella. The expression of these proteins is ubiquitous in plant tissues and primarily localises in the cell nucleus. HMGB proteins are involved in several nuclear processes, but the role of ARID-HMG proteins in plants remains poorly explored. Here, we performed DNA-protein interaction studies with Arabidopsis ARID-HMG protein HMGB11 (At1g55650) to understand the functionality of this protein and its individual domains. DNA binding assays revealed that AtHMGB11 can bind double-stranded DNA with a weaker affinity (Kd = 475 ± 17.9 nM) compared to Arabidopsis HMGB1 protein (Kd = 39.8 ± 2.68 nM). AtHMGB11 also prefers AT-rich DNA as a substrate and shows structural bias for supercoiled DNA. Molecular docking of the DNA-AtHMGB11 complex indicated that the protein interacts with the DNA major groove, mainly through its ARID domain and the junction region connecting the ARID and the HMG-box domain. Also, predicted by the docking model, mutation of Lys(85) from the ARID domain and Arg(199) & Lys(202) from the junction region affects the DNA binding affinity of AtHMGB11. In addition, AtHMGB11 and its truncated form containing the HMG-box domain can not only promote DNA mini-circle formation but are also capable of inducing negative supercoils into relaxed plasmid DNA suggesting the involvement of this protein in several nuclear events. Overall, the study signifies that both the ARID and the HMG-box domain contribute to the optimal functioning of ARID-HMG protein in vivo.
ARID-HMG 蛋白是一类新的 HMG 盒蛋白家族，其 AT 丰富的相互作用结构域（ARID）与 HMG 盒结构域融合在单个多肽链中。ARID-HMG 蛋白在植物中具有高度特异性，在开花植物和苔藓植物（如 Physcomitrella）中都发现了同源物。这些蛋白在植物组织中广泛表达，主要定位于细胞核内。HMGB 蛋白参与了多种核过程，但 ARID-HMG 蛋白在植物中的作用仍未得到充分探索。在这里，我们对拟南芥 ARID-HMG 蛋白 HMGB11（At1g55650）进行了 DNA-蛋白相互作用研究，以了解该蛋白及其各个结构域的功能。DNA 结合实验表明，与拟南芥 HMGB1 蛋白（Kd=39.8±2.68 nM）相比，AtHMGB11 可以较弱的亲和力（Kd=475±17.9 nM）结合双链 DNA。AtHMGB11 还优先作为底物与富含 AT 的 DNA 结合，并表现出对超螺旋 DNA 的结构偏好。DNA-AtHMGB11 复合物的分子对接表明，该蛋白与 DNA 的大沟相互作用，主要通过其 ARID 结构域和连接 ARID 与 HMG 盒结构域的连接区。此外，根据对接模型预测，ARID 结构域中的 Lys(85)和连接区中的 Arg(199)和 Lys(202)的突变会影响 AtHMGB11 的 DNA 结合亲和力。此外，AtHMGB11 及其包含 HMG 盒结构域的截断形式不仅可以促进 DNA 小环的形成，还可以将负超螺旋诱导到松弛的质粒 DNA 中，这表明该蛋白参与了几种核事件。总的来说，该研究表明，ARID 和 HMG 盒结构域都有助于 ARID-HMG 蛋白在体内的最佳功能。
