Elongation of Helix III of the NK-2 Homeodomain upon Binding to DNA: A Secondary Structure Study by NMR

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Abstract

The secondary structure of the homeodomain encoded by the NK-2 gene from Drosophila melanogaster, in both the free and DNA-bound states, was determined in solution using two- and three-dimensional (2D and 3D) NMR spectroscopy. Proton and 15N studies were carried out on a 77 amino acid residue protein that contains the homeodomain, which was synthesized in Escherichia coli. On the basis of NOE connectivities, vicinal coupling constants, and proton-deuterium exchange behavior, three helical segments were found that consist of homeodomain amino acid residues 10-22, 28-38, and 42-52 for the protein in the absence of DNA. The major structural differences between free NK-2 and other homeodomains are the increased internal mobility of the second helix and the shorter length of the third helix, also termed the recognition helix. Despite this shorter helix, NK-2 exhibits high-affinity binding to DNA compared to other homeodomains (kD = 2.0 x 10(-10) M; L.-H. Wang and M. Nirenberg, unpublished results). The formation of the complex of NK-2 with the duplex DNA (TGTGTCAAGTG-GCTGT) significantly increases the thermal stability of the protein. The Tm increases from 25 degrees C (free NK-2) to > 47 degrees C (DNA-bound NK-2). Also, a dramatic increase in the length of helix III is observed. In the absence of DNA, the DNA recognition helix is 11 amino acid residues long (residues 42-52), whereas in the presence of DNA, the length of this helix extends to 19 amino acids (residues 42-60).(ABSTRACT TRUNCATED AT 250 WORDS)

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