N-terminal basic residues of HOXD9 homeodomain drives its target-site searching along DNA

HOXD9 is a representative HOX-family transcription factor that binds specific promoters to determine the somite differentiation. Aberrations in HOXD9 expression are associated with diseases like tumorigenesis and metastasis. Currently, an atomic-level understanding of the molecular mechanism underlying the target-searching dynamics of HOXD9 along DNA remains elusive, and the key regulatory motifs that dictate the HOXD9 diffusion along DNA and HOXD9-DNA interplays are also unknown. Here, through constructing kinetic models based on extensive all-atom molecular dynamics simulations, we reveal the complete target-searching dynamics of the HOXD9 homeodomain along a 10-bp DNA segment. We found the N-terminal arm is directly involved in driving the HOXD9 diffusion along DNA before targeting to the specific-site. Particularly, several positively-charged residues, including R2-R5, play critical roles in forming direct contacts with DNA, mainly via electrostatic interactions. As approaches to the target-site, HOXD9 tends to plug its N-terminus out of the minor-groove, meanwhile, establishes specific recognitions with the specific-site via R5 and the helix-3 domain. Further site-directed mutagenetic assays validated the predicted functional roles of these N-terminal residues. Phylogenetic tree analyses of 154 HOX proteins indicate highly conserved structural features in the N-terminus, suggesting the proposed target-searching mechanism for HOXD9 could be generalized to other homeodomain proteins.