STRUCTURAL BASIS OF AN EMBRYONICALLY LETHAL SINGLE ALA-]THR MUTATION IN THE VND NK-2 HOMEODOMAIN/

The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue a lanine to threonine replacement in position 35 of the homeodomain. Mul tidimensional nuclear magnetic resonance, circular dichroism, and elec trophoretic gel retardation assays were carried out on recombinant 80- aa residue proteins that encompass the wildtype and mutant homeodomain s. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded co nformation free in solution at temperatures down to -5 degrees C in co ntrast to the behavior of the corresponding wild-type vnd/NK-2 homeodo main, which is folded into a functional three-dimensional structure be low 25 degrees C. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain, Although the three-dimensional stru cture of the mutant A35T vnd/NK-2 in the DNA bound state shows charact eristic helix-turn-helix behavior similar to that of the wild-type hom eodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen o f residue 35. In the A35T mutant this amide proton resonance is shifte d upfield by 1.27 ppm relative to the resonance frequency for the wild -type analog, thereby indicating a significant alteration of this i,i- 5 hydrogen bond.