Molecular insights into the binding dynamics of transcription factor TBR1 to T-box DNA sequences

TBR1 is a transcription factor critical for brain development that recognises a specific nucleotide sequence, the T-box binding element (TBE). Dysregulation or mutation of TBR1 is linked to a spectrum of neurodevelopmental disorders, including autism, speech delay, and intellectual disability, yet the molecular mechanism underlying its DNA recognition remains poorly defined. Here, we examine how the TBR1 T-box domain recognises DNA containing either a single TBE or a palindromic arrangement of two adjacent TBEs. Both sequences are derived from naturally occurring TBR1 binding motifs located within genomic regions annotated as candidate enhancers of autism-associated genes. The TBR1 T-box binds its cognate element in a sequence-specific, enthalpically driven, and entropically opposed manner. Single-molecule Förster resonance energy transfer (smFRET) reveals that the single-site sequence binds one monomer, whereas the palindromic sequence can recruit a second monomer and exhibits additional dynamic events unique to this architecture. These include short-range transitions of a monomer along the palindromic DNA before dissociation or before the arrival of a second monomer. Molecular docking and dynamics simulations support these observations, predicting fewer stabilising contacts per TBE and hence greater conformational flexibility for the palindromic complex. Together, our findings reveal how DNA architecture modulates affinity and kinetics within the T-box family: single TBEs promote stable monomeric binding, whereas palindromic arrangements enable dual occupancy and dynamic exchange. This study provides the first detailed mechanistic insight into DNA recognition by a T-box transcription factor, advancing understanding of its role in neurodevelopmental gene regulation.