Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, Michigan, USA.
Department of Chemistry, Michigan State University, East Lansing, Michigan, USA.
J Biol Chem. 2021 Jan-Jun;296:100708. doi: 10.1016/j.jbc.2021.100708. Epub 2021 Apr 24.
About a third of the plant basic helix-loop-helix (bHLH) transcription factors harbor a C-terminal aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain, which was originally identified in the maize R regulator of anthocyanin biosynthesis, where it modulates the ability of the bHLH to dimerize and bind DNA. Characterization of other bHLH ACT-like domains, such as the one in the Arabidopsis R ortholog, GL3, has not definitively confirmed dimerization, raising the question of the overall role of this potential regulatory domain. To learn more, we compared the dimerization of the ACT-like domains of R (R) and GL3 (GL3). We show that R dimerizes with a dissociation constant around 100 nM, over an order of magnitude stronger than GL3. Structural predictions combined with mutational analyses demonstrated that V568, located in a hydrophobic pocket in R, is important: when mutated to the Ser residue present in GL3, dimerization affinity dropped by almost an order of magnitude. The converse S595V mutation in GL3 significantly increased the dimerization strength. We cloned and assayed dimerization for all identified maize ACT-like domains and determined that 12 of 42 formed heterodimers in yeast two-hybrid assays, irrespective of whether they harbored V568, which was often replaced by other aliphatic amino acids. Moreover, we determined that the presence of polar residues at that position occurs only in a small subset of anthocyanin regulators. The combined results provide new insights into possibly regulatory mechanisms and suggest that many of the other plant ACT-like domains associate to modulate fundamental cellular processes.
大约三分之一的植物碱性螺旋-环-螺旋（bHLH）转录因子在 C 端含有天冬氨酸激酶、分支酸变位酶和 TyrA（ACT）样结构域，该结构域最初在玉米 R 调控花青素生物合成中被发现，在该结构域中调节 bHLH 二聚化和结合 DNA 的能力。对其他 bHLH ACT 样结构域的特征，如拟南芥 R 同源物 GL3 中的 ACT 样结构域，尚未明确证实二聚化，这引发了对该潜在调节结构域的整体作用的质疑。为了更多地了解这一点，我们比较了 R（R）和 GL3（GL3）的 ACT 样结构域的二聚化。我们表明，R 以约 100 nM 的解离常数二聚化，比 GL3 强一个数量级。结构预测结合突变分析表明，位于 R 中疏水性口袋中的 V568 很重要：当突变为 GL3 中存在的 Ser 残基时，二聚化亲和力下降了近一个数量级。GL3 中的反向 S595V 突变显著增加了二聚化强度。我们克隆并检测了所有鉴定出的玉米 ACT 样结构域的二聚化，确定了 42 个中的 12 个在酵母双杂交测定中形成异二聚体，无论它们是否含有 V568，V568 通常被其他脂族氨基酸取代。此外，我们确定该位置的极性残基的存在仅发生在一小部分花青素调控因子中。综合结果为可能的调节机制提供了新的见解，并表明许多其他植物 ACT 样结构域的存在是为了调节基本的细胞过程。
