THE 3-DIMENSIONAL SOLUTION STRUCTURE OF THE NK-2 HOMEODOMAIN FROM DROSOPHILA

We describe the NMR determination of the three-dimensional structure o f a 77 amino acid residue protein, which consists of the 60 residue NK -2 homeodomain from Drosophila melanognster and adjacent amino acid re sidues. The NK-2 homeodomain protein is part of a 723 amino acid resid ue protein which is expressed early in embryonic development in part o f the central nervous system. NK-2 was characterized using both a natu ral abundance and a uniformly N-15 enriched sample by two-dimensional and three-dimensional NMR experiments. The average root-mean-square de viation for 30 structures for residues 8 to 53 is 0.40 Angstrom for th e backbone heavy-atoms and 0.72 Angstrom for the backbone and side-cha in heavy-atoms. These structures were obtained from 986 NOE-derived up per and lower bound restraints. The three-dimensional structure contai ns three helices which consist of homeodomain amino acid residues 10 t o 22, 28 to 38 and 42 to 52, as well as a turn between helix II and II I, characteristic of homeodomains. Residues 53 to 60 of the DNA recogn ition helix are not fully ordered in the absence of DNA. In the free s tate this segment adopts a flexible but helix-like structure between r esidues 53 and 56 and is disordered from residues 57 to 60 although, a s shown previously, the helix elongates by eight residues upon binding to DNA. The role of variable residues 52, 54 and 56 in determining th e structure and flexibility of the recognition helix, as well as the s tability of the NK-2 homeodomain as manifested by its thermal denatura tion, are discussed. (C) 1995 Academic Press Limited