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Fine-tuning of intrinsic N-Oct-3 POU domain allostery by regulatory DNA targets.

Alazard R, Mourey L, Ebel C, Konarev PV, Petoukhov MV, Svergun DI, Erard M - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institut de Pharmacologie et de Biologie Structurale, 205 Route de Narbonne, 31077 Toulouse, France.

ABSTRACT
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.

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Distance distribution functions of the free N-Oct-3 DBD (green), the free CRH DNA (magenta), the N-Oct-3/CRH (red) and the N-Oct-3/DRα (blue) complexes.
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Figure 8: Distance distribution functions of the free N-Oct-3 DBD (green), the free CRH DNA (magenta), the N-Oct-3/CRH (red) and the N-Oct-3/DRα (blue) complexes.

Mentions: Processed X-ray scattering patterns corresponding to the free N-Oct-3 DBD are presented in Figure 7A and B (data groups 1), alongside those from the free DNA fragments (data groups 2) and from the equimolecular N-Oct-3 DBD/DNA complexes (data groups 3). The structural parameters computed from the experimental data, including the radius of gyration (Rg) and maximum particle dimension (Dmax), are displayed in Table 1. The estimated effective mass (Meff) of the free N-Oct-3 DBD agrees within experimental error with the value expected from the sequence (Mseq), confirming that the protein is monomeric in solution. The distance distribution functions computed from the experimental data (Figure 8) emphasize the elongated shape of the free form(s), and the similarities between the gyration radii of the free N-Oct-3 DBD and of its complexes with each promoter DNA fragment. Note the good agreement between the free N-Oct-3 DBD gyration and hydrodynamic radii.Figure 7.


Fine-tuning of intrinsic N-Oct-3 POU domain allostery by regulatory DNA targets.

Alazard R, Mourey L, Ebel C, Konarev PV, Petoukhov MV, Svergun DI, Erard M - Nucleic Acids Res. (2007)

Distance distribution functions of the free N-Oct-3 DBD (green), the free CRH DNA (magenta), the N-Oct-3/CRH (red) and the N-Oct-3/DRα (blue) complexes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935007&req=5

Figure 8: Distance distribution functions of the free N-Oct-3 DBD (green), the free CRH DNA (magenta), the N-Oct-3/CRH (red) and the N-Oct-3/DRα (blue) complexes.
Mentions: Processed X-ray scattering patterns corresponding to the free N-Oct-3 DBD are presented in Figure 7A and B (data groups 1), alongside those from the free DNA fragments (data groups 2) and from the equimolecular N-Oct-3 DBD/DNA complexes (data groups 3). The structural parameters computed from the experimental data, including the radius of gyration (Rg) and maximum particle dimension (Dmax), are displayed in Table 1. The estimated effective mass (Meff) of the free N-Oct-3 DBD agrees within experimental error with the value expected from the sequence (Mseq), confirming that the protein is monomeric in solution. The distance distribution functions computed from the experimental data (Figure 8) emphasize the elongated shape of the free form(s), and the similarities between the gyration radii of the free N-Oct-3 DBD and of its complexes with each promoter DNA fragment. Note the good agreement between the free N-Oct-3 DBD gyration and hydrodynamic radii.Figure 7.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institut de Pharmacologie et de Biologie Structurale, 205 Route de Narbonne, 31077 Toulouse, France.

ABSTRACT
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.

Show MeSH