Specific binding between Arabidopsis thaliana phytochrome-interacting factor 3 (AtPIF3) bHLH and G-box originated prior to embryophyte emergence

• Published in: BMC plant biology Vol. 24; no. 1; p. 1060
• Main Authors: Hsin, Kuan-Ting, Lee, Yu-Hsuan, Lin, Kai-Chun, Chen, Wei, Cheng, Yi-Sheng
• Format: Journal Article
• Language: English
• Published: London BioMed Central 11.11.2024; BioMed Central Ltd; BMC
• Subjects: Agriculture; Amino Acid Sequence; Amino acids; Analysis; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Basic Helix-Loop-Helix Transcription Factors - genetics; Basic Helix-Loop-Helix Transcription Factors - metabolism; Binding; Biological evolution; Biomedical and Life Sciences; Calorimetry; Circadian clock; Circadian rhythm; Conservation; Control; Deoxyribonucleic acid; DNA; DNA binding preference; domain; Electrophoretic mobility; evolution; Evolution, Molecular; Evolutionary conservation; Flowers & plants; gel electrophoresis; Gene expression; Genetic aspects; Genomes; Helix-loop-helix proteins (basic); hydrogen bonding; Hydrogen bonds; Identification and classification; Life Sciences; Light; Methods; Phytochrome; Phytochrome-interacting factor 3; Plant Sciences; point mutation; Preferences; Protein Binding; Protein-DNA interaction; Proteins; recombinant proteins; Residues; Signal transduction; species; Titration; Titration calorimetry; Tree Biology; Wildlife conservation; Taiwan; Circadian clock; DNA binding preference; Protein-DNA interaction; Conservation
• ISSN: 1471-2229; 1471-2229
• DOI: 10.1186/s12870-024-05777-z

The 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.