Kuan-Ting HsinYu-Hsuan LeeKai-Chun LinWei ChenYI-SHENG CHENG2024-12-032024-12-032024-11-11https://www.scopus.com/record/display.uri?eid=2-s2.0-85209484122&origin=resultslisthttps://scholars.lib.ntu.edu.tw/handle/123456789/723492Article number 1060The basic helix-loop-helix (bHLH) domain via critical amino acid residues on basic region binding to E-box (5′-CANNTG-3′) is known in embryophyte. However, the dictated E-box types selection by bHLH dimers and the significant impact of these critical amino acid residues along embryophyte evolution remain unclear. The Arabidopsis thaliana PIF3-bHLH (AtPIF3-bHLH) recombinant protein and a series of AtPIF3-bHLH mutants were synthesized and analyzed. The reduced DNA binding ability and affinity of AtPIF3-bHLH point-mutation proteins, observed via fluorescence-based electrophoretic mobility shift assay (fEMSA) and isothermal titration calorimetry (ITC), suggest the critical role of these DNA-recognition sites in maintaining the AtPIF3-bHLH–DNA interaction. The purifying selection signals and the DNA-recognition-site conservation at the species level suggest the invariant roles of these sites throughout embryophyte evolution. The G-box outcompeted other types of E-box for binding in our competitive fEMSAs. The dynamic hydrogen bond formed between AtPIF3-bHLH and the G-box core indicates flexible identification of the core region. These features highlight a fast fixation of the bHLH-G-box recognition mechanism through embryophyte evolution and serve as a blueprint for studying DNA recognition determinants of other TF families.entrueCircadian clockConservationDNA binding preferenceProtein-DNA interaction[SDGs]SDG2[SDGs]SDG13[SDGs]SDG15journal article10.1186/s12870-024-05777-z2-s2.0-85209484122