Structural Insights into the Recognition of the A/T-rich Motif in Target Gene Promoters by the LMX1a Homeobox Domain.

LIM homeodomain transcription factor 1‐alpha (LMX1a) is a neuronal lineage‐specific transcription activator that plays an essential role during the development of midbrain dopaminergic (mDA) neurons. LMX1a induces the expression of multiple key genes, which ultimately determine the morphology, physiology, and functional identity of mDA neurons. This function of LMX1a is dependent on its homeobox domain. Here, we determined the structures of the LMX1a homeobox domain in complex with the promoter sequences of the Wnt family member 1 (WNT1) or paired like homeodomain 3 (Pitx3) gene, respectively. The complex structures revealed that the LMX1a homeobox domain employed its α3 helix and an N‐terminal loop to achieve specific target recognition. The N‐terminal loop (loop1) interacted with the minor groove of the double‐stranded DNA (dsDNA), whereas the third α‐helix (α3) was tightly packed into the major groove of the dsDNA. Structure‐based mutations in the α3 helix of the homeobox domain significantly reduced the binding affinity of LMX1a to dsDNA. Moreover, we identified a nonsyndromic hearing loss (NSHL)‐related mutation, R199, which yielded a more flexible loop and disturbed the recognition in the minor groove of dsDNA, consistent with the molecular dynamics (MD) simulations. Furthermore, overexpression of Lmx1a promoted the differentiation of SH‐SY5Y cells and upregulated the transcription of WNT1 and PITX3 genes. Hence, our work provides a detailed elucidation of the specific recognition between the LMX1a homeobox domain and its specific dsDNA targets, which represents valuable information for future investigations of the functional pathways that are controlled by LMX1a during mDA neuron development.