Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA.
Eight of the nine homeobox genes of the Hoxb locus encode proteins which 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 the Hox-Pbx1a-DNA complexes vary >100-fold, with the proteins from the middle of the locus (Hoxb-5 and Hoxb-6) forming very stable complexes, while Hoxb-4, Hoxb-7 and Hoxb-8 form complexes of intermediate stability and proteins at the 3'-side of the locus (Hoxb-1-Hoxb-3) form complexes which are very unstable. Although Hox-b proteins containing longer linker sequences between the hexapeptide and homeodomains formed unstable complexes, shortening the linker did not confer complex stability. Homeodomain swapping experiments revealed that this motif does not independently determine complex stability. Naturally occurring variations within the hexapeptides of specific Hox proteins also do not explain complex stability differences. However, two core amino acids (tryptophan and methionine) which are absolutely conserved within the hexapeptide 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 different flanking regions, form complexes of similar stability. These data 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.
Shen
W F
WF
Department of Medicine San Francisco Veterans Affairs Medical Center, CA 94121, USA.
Chang
C P
CP
Rozenfeld
S
S
Sauvageau
G
G
Humphries
R K
RK
Lu
M
M
Lawrence
H J
HJ
Cleary
M L
ML
Largman
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D017421:Sequence Analysis
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D046911:Macromolecular Substances; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins; C083400:PBX1 protein, human; D004247:DNA