INTERACTIONS OF THE VND NK-2 HOMEODOMAIN WITH DNA BY NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY - BASIS OF BINDING-SPECIFICITY/

The interactions responsible for the nucleotide sequence-specific bind ing of the vnd/NK-2 homeodomain of Drosophila melanogaster to its cons ensus DNA binding site have been identified. A three-dimensional struc ture of the vnd/NK-2 homeodomain-DNA complex is presented, with emphas is on the structure of regions of observed protein-DNA contacts. This structure is based on protein-DNA distance restraints derived from NMR data, along with homology modeling, solvated molecular dynamics, and results from methylation and ethylation interference experiments. Heli x III of the homeodomain binds in the major groove of the DNA and the N-terminal arm binds in the minor groove, in analogy with other homeod omain-DNA complexes whose structures have been reported. The vnd/NK-2 homeodomain recognizes the unusual DNA consensus sequence 5'-CAAGTG-3' . The roles in sequence specificity and strength of binding of individ ual amino acid residues that make contact with the DNA are described. We show, based primarily on the observed protein-DNA contacts, that th e interaction of Y54 with the DNA is the major determinant of this unc ommon nucleotide binding specificity in the vnd/NK-2 homeodomain-DNA c omplex.