A hydrophobic residue stabilizes dimers of regulatory ACT-like domains in plant basic helix-loop-helix transcription factors.

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.