The three-dimensional structure of the vnd/NK-2 homeodomain bound to an uncommon18 base pair DNA consensus segment that contains 5'-CAAGTG-3' has been analyzed by NMR spectroscopy. The interactions responsible for the nucleotide sequence-specific binding have been identified, where tyrosine in position 54 previously was shown to be the major determinant of the unusual consensus nucleotide sequence. Mutational analysis was carried out on four positions (35, 52, 54, and 56) in the homeodomain. Mutation of hysitidine to arginine in position 52 of the homeodomain results in an increase of 4 - 7 degrees in the thermal denaturation temperature of the homeodomain. This observation correlates well with temperature dependent developmental anomalies associated with corresponding mutants found in vivo. Mutation of tyrosine to methionine in position 54 results in a decrease of one order of magnitude in the binding affinity of the homeodomain for the DNA. Resonance assignments for the 54 mutation in the free and DNA bound states have been made. The structure of the mutation protein free in solution has been determined. Contacts between the mutant homeodomain and the DNA have been identified and the three-dimensional structure of the corresponding DNA complex is in the process of being determined. The structure determination process will take advantage of new pulse sequence experiments, as well as the measurement of relaxation times, motional anisotropies, and chemical shift anisotropies to provide the highest quality structures. An 80 amino acid residue protein containing the vnd/NK-2 homeodomain with a single site mutation of alanine to threonine in position 35 of the homeodomain has been expressed and purified. This mutant of protein is of particular interest because significant neuronal hypoplasia and early embryonic lethality in Drosophila melanogaster is has been mapped to a single base modification in the vnd/NK-2 homeobox gene that codes for threonine in place of the highly conserved alanine in position 35 of the homeodomain. In contrast to the native or wild type vnd/NK-2, which undergoes a reversible thermal denaturation transition at 25 degrees centrigrade, the threonine mutant vnd/NK-2 homeodomain is unstructured at all accessible temperatures. Nevertheless, the mutant homeodomain recognizes and binds tightly to the consensus DNA sequence with many homeodomain-DNA contacts that are found in the native homeodomain. The determination of the three-dimensional structure of the homeodomain-DNA complex and a precise comparison of the DNA affinity constants is in progress. Experiments to determine the role of water molecules at the protein-DNA interface on sequence specific binding has been initiated. NMR pulse sequences have been developed and optimized to study the interactions between water and the homeodomain or the DNA.
Showing the most recent 10 out of 26 publications
