The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions.
The transcription factor Pax6 is essential for the development of the eyes and the central nervous system of vertebrates and invertebrates. Pax6 contains two DNA-binding domains; an N-terminal paired domain and a centrally located homeodomain. We have previously shown that the vertebrate paired-less isoform of Pax6 (Pax6DeltaPD), and several other homeodomain proteins, interact with the full-length isoform of Pax6 enhancing Pax6-mediated transactivation from paired domain-DNA binding sites. By mutation analyses and molecular modeling we now demonstrate that, surprisingly, the recognition helix for specific DNA binding of the homeodomains of Pax6 and Chx10 interacts with the C-terminal RED subdomain of the paired domain of Pax6. Basic residues in the recognition helix and the N-terminal arm of the homeodomain form an interaction surface that binds to an acidic patch involving residues in helices 1 and 2 of the RED subdomain. We used fluorescence resonance energy transfer assays to demonstrate such interactions between Pax6 molecules in the nuclei of living cells. Interestingly, two mutations in the homeodomain recognition helix, R57A and R58A, reduced protein-protein interactions, but not DNA binding of Pax6DeltaPD. These findings suggest a critical role for the recognition helix and N-terminal arm of the paired class homeodomain in protein-protein interactions.
Bruun
Jack-Ansgar
JA
Biochemistry Department, Institute of Medical Biology, University of Tromsø 9037 Tromsø, Norway.
Thomassen
Ernst Ivan Simon
EI
Kristiansen
Kurt
K
Tylden
Garth
G
Holm
Turid
T
Mikkola
Ingvild
I
Bjørkøy
Geir
G
Johansen
Terje
T
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004252:DNA Mutational Analysis; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D000596:Amino Acids; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D004247:DNA; D001120:Arginine