HOX HOMEODOMAIN PROTEINS EXHIBIT SELECTIVE COMPLEX STABILITIES WITH PBX AND DNA

Eight of the nine homeobox genes of the Herb locus encode proteins whi ch contain a conserved hexapeptide motif upstream from the homeodomain . All eight proteins (Hoxb-1-Hoxb-8) bind to a target oligonucleotide in the presence of Pbx1a under conditions where minimal or no binding is detected for the Hox or Pbx1a proteins alone. The stabilities of th e Hox-Pbx1a-DNA complexes vary >100-fold, with the proteins from the m iddle of the locus (Hoxb-1 and Hoxb-6) forming very stable complexes, while Hoxb-4, Hoxb-7 and Hoxb-8 form complexes of intermediate stabili ty and proteins at the 3'-side of the locus (Hoxb-1-Hoxb-9) form compl exes which are very unstable. Although Hox-b proteins containing longe r linker sequences between the hexapeptide and homeodomains formed uns table complexes, shortening the linker did not confer complex stabilit y. Homeodomain swapping experiments revealed that this motif does not independently determine complex stability. Naturally occurring variati ons within the hexapeptides of specific Hox proteins also do not expla in complex stability differences. However, two core amino acids (trypt ophan and methionine) which are absolutely conserved within the hexape ptide domains appear to be required for complex formation. Removal of N- and C-terminal flanking regions did not influence complex stability and the members of paralog group 4 (Hoxa-4, b-4, c-4 and d-4), which share highly conserved hexapeptides, linkers and homeodomains but diff erent flanking regions, form complexes of similar stability. These dat a suggest that the structural features of Hox proteins which determine Hox-Pbx1a-DNA complex stability reside within the precise structural relationships between the homeodomain, hexapeptide and linker regions.