Autism-associated substitutions at ASN374 disrupt forkhead box P2-mediated regulation of DNA binding by t-box brain protein 1.

TBR1 is a neurodevelopmental transcription factor whose function depends on the coordinated integration of DNA binding with regulatory protein-protein interactions. Despite physiological links between TBR1 and other transcription factors, the biochemical mechanisms that couple DNA binding to partner-mediated regulation, and how these processes are disrupted by disease-associated variants, remain poorly defined. Here we provide the first direct biochemical evidence that the FOXP2 forkhead domain (FHD) binds the TBR1 T-box domain and we show that this interaction is modulated by residue Asn374. Using fluorescence anisotropy and EMSA, we demonstrate that autism-associated N374H and N374Y substitutions weaken TBR1 binding to T-box DNA and alter its response to FOXP2. Although both variants reduce FOXP2-TBR1 affinity, they paradoxically resist FOXP2-driven dissociation of TBR1 from DNA, indicating impaired FOXP2-dependent regulation of TBR1. Molecular modelling and molecular dynamics simulations indicate that substitutions at Asn374 do not abolish the FOXP2-binding surface but instead reconfigure neighbouring contacts and alter conformational dynamics, yielding a more compact yet flexible T-box domain within FOXP2-associated complexes. Collectively, our results identify Asn374 as an allosteric control point that coordinates TBR1 DNA-binding dynamics with FOXP2 engagement. This work provides a mechanistic framework for how FOXP2-dependent modulation of transcription during neurodevelopment may be altered in autism.