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 sit e H52R, Y54M, and T56W mutations, two double site H52R/T56W and Y54M/T 56W mutations, and one triple site H52R/Y54M/T56W mutation were invest igated. These positions were chosen based on their variability across homeodomains displaying differences in secondary structure and DNA bin ding specificity. Multidimensional NMR, electrophoretic mobility shift assays, and circular dichroism spectropolarimetry studies were carrie d out on recombinant 80-amino acid residue proteins containing the hom eodomain, Position 56, but more importantly position 56 in combination with position 52, plays an important role in determining the length o f the recognition helix. The H52R mutation alone does not affect the l ength of this helix but does increase the thermal stability. introduct ion of site mutations at positions 52 and 56 in vnd/NK-2 does not modi fy their high affinity binding to the 18-base pair DNA fragment contai ning the vnd/NK-2 consensus binding sequence, CAAGTG, Site mutations i nvolving 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 interact ions are described.