Dynamic Local Polymorphisms in the Gbx1 Homeodomain Induced by DNA Binding

The Gastrulation Brain Homeobox 1 (Gbx1) gene encodes the Gbx1 homeodomain that targets TAATTA motifs in double-stranded DNA (dsDNA). Residues Glu17 and Arg52 in Gbx1 form a salt bridge, which is preserved in crystal structures and molecular dynamics simulations of homologous homeodomain-DNA complexes. In contrast, our nuclear magnetic resonance (NMR) studies show that DNA binding to Gbx1 induces dynamic local polymorphisms, which include breaking of the Glu17-Arg52 salt bridge. To study this interaction, we produced a variant with Glu17Arg and Arg52Glu mutations, which exhibited the same fold as the wild-type protein, but a 2-fold reduction in affinity for dsDNA. Analysis of the NMR structures of the Gbx1 homeodomain in the free form, the Gbx1[E17R,R52E] variant, and a Gbx1 homeodomain-DNA complex showed that stabilizing interactions of the Arg52 side chain with the DNA backbone are facilitated by transient breakage of the Glu17-Arg52 salt bridge in the DNA-bound Gbx1.