Probing the role of the protonation state of a minor groove-linker histidine in Exd-Hox-DNA binding

DNA recognition and targeting by transcription factors (TFs) through specific binding are fundamental in biological processes. Furthermore, the histidine protonation state at the TF-DNA binding interface can significantly influence the binding mechanism of TF-DNA complexes. Nevertheless, the role of histidine in TF-DNA complexes remains underexplored. Here, we employed all-atom molecular dynamics simulations using AlphaFold2-modeled complexes based on previously solved co-crystal structures to probe the role of the His-12 residue in the Extradenticle (Exd)-Sex combs reduced (Scr)-DNA complex when binding to Scr and Ultrabithorax (Ubx) target sites. Our results demonstrate that the protonation state of histidine notably affected the DNA minor-groove width profile and binding free energy. Examining flanking sequences of various binding affinities derived from SELEX-seq experiments, we analyzed the relationship between binding affinity and specificity. We uncovered how histidine protonation leads to increased binding affinity but can lower specificity. Our findings provide new mechanistic insights into the role of histidine in modulating TF-DNA binding. SIGNIFICANCE This study reveals the molecular mechanism for the significance of a histidine residue in the Scr Hox protein. Our findings indicate that His-12 plays a crucial role in stabilizing the linker region of the Hox protein with the Scr core motif by regulating the minor groove shape profile, which is essential for Hox specificity. The uniqueness of His-12 lies in its ability to adopt two protonation states at biological pH. Our results demonstrate that the differences in behavior between the two protonation states of His-12 not only explain its preference toward different core motifs but also provide insights into its preference for binding of high- and low-affinity target sequences.