Fine-tuning of intrinsic N-Oct-3 POU domain allostery by regulatory DNA targets.
The 'POU' (acronym of Pit-1, Oct-1, Unc-86) family of transcription factors share a common DNA-binding domain of approximately 160 residues, comprising so-called 'POUs' and 'POUh' sub-domains connected by a flexible linker. The importance of POU proteins as developmental regulators and tumor-promoting agents is due to linker flexibility, which allows them to adapt to a considerable variety of DNA targets. However, because of this flexibility, it has not been possible to determine the Oct-1/Pit-1 linker structure in crystallographic POU/DNA complexes. We have previously shown that the neuronal POU protein N-Oct-3 linker contains a structured region. Here, we have used a combination of hydrodynamic methods, DNA footprinting experiments, molecular modeling and small angle X-ray scattering to (i) structurally interpret the N-Oct-3-binding site within the HLA DRalpha gene promoter and deduce from this a novel POU domain allosteric conformation and (ii) analyze the molecular mechanisms involved in conformational transitions. We conclude that there might exist a continuum running from free to 'pre-bound' N-Oct-3 POU conformations and that regulatory DNA regions likely select pre-existing conformers, in addition to molding the appropriate DBD structure. Finally, we suggest that a specific pair of glycine residues in the linker might act as a major conformational switch.
Alazard
Robert
R
Institut de Pharmacologie et de Biologie Structurale, 205 Route de Narbonne, 31077 Toulouse, France.
Mourey
Lionel
L
Ebel
Christine
C
Konarev
Peter V
PV
Petoukhov
Maxim V
MV
Svergun
Dmitri I
DI
Erard
Monique
M
D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D018983:DNA Footprinting; D005998:Glycine / Q000737:chemistry; D006684:HLA-DR Antigens / Q000235:genetics; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D050809:POU Domain Factors / Q000737:chemistry* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D051381:Rats; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D006684:HLA-DR Antigens; D018398:Homeodomain Proteins; D050809:POU Domain Factors; C085076:transcription factor Brn-2; D005998:Glycine