Structural insight into the interaction between the Hox and HMGB1 and understanding of the HMGB1-enhancing effect of Hox-DNA binding

• The solution structure of Hox homeodomain including N-terminal arm.
• The residue-specific and thermodynamic analysis of the interaction between Hox, HMGB1, and DNA were performed.
• The probable mechanism that HMGB1 facilitates the Hox-binding to its target DNA was suggested.

The Hox DNA binding domain, the homeodomain, plays critical roles in genetic control of development and cell fate determination. The variable regulatory functions of Hox proteins are accomplished by binding to target DNA sequences and collaborating protein partners that includes human high mobility group B1 (HMGB1). To better understand the interaction between Hox and HMGB1 and the facilitation of Hox-DNA binding by HMGB1, we solved the solution structure of the homeodomain of Hox including the N-terminal arm region (Hoxc9DBD hereafter). In addition, the details of the interaction between these two proteins, as well as DNA binding of the Hox–HMGB1 complex, were investigated by NMR, ITC, and EMSA. The results suggest that binding of the HMGB1 A-box to Hoxc9DBD makes the loop-1 (loop preceding helix-2 of Hoxc9DBD) more access to DNA backbone, which facilitate Hox-DNA binding with enhanced affinity.