Site-directed mutations in the vnd/NK-2 homeodomain. Basis of variations in structure and sequence-specific DNA binding

Secondary structures, DNA binding properties, and thermal denaturation behavior of six site-directed mutant homeodomains encoded by the vnd/NK-2 gene from Drosophila melanogaster are described. Three single site H52R, Y54M, and T56W mutations, two double site H52R/T56W and Y54M/T56W mutations, and one triple site H52R/Y54M/T56W mutation were investigated. These positions were chosen based on their variability across homeodomains displaying differences in secondary structure and DNA binding specificity. Multidimensional NMR, electrophoretic mobility shift assays, and circular dichroism spectropolarimetry studies were carried out on recombinant 80-amino acid residue proteins containing the homeodomain. Position 56, but more importantly position 56 in combination with position 52, plays an important role in determining the length of the recognition helix. The H52R mutation alone does not affect the length of this helix but does increase the thermal stability. Introduction of site mutations at positions 52 and 56 in vnd/NK-2 does not modify their high affinity binding to the 18-base pair DNA fragment containing the vnd/NK-2 consensus binding sequence, CAAGTG. Site mutations involving position 54 (Y54M, Y54M/T56W, and H52R/Y54M/T56W) all show a decrease of 1 order of magnitude in their binding affinity. The roles in structure and sequence specificity of individual atom-atom interactions are described.