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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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Characterization of the N-Oct-3 POU domain. (A) Detection of a single band with the expected N-Oct-3 DBD molecular mass by Coomassie-blue staining in 13% SDS-PAGE (see the molecular mass markers on the right). (B) Dynamic light scattering of the N-Oct-3 DBD (see text). (C) Calibration curve obtained by FPLC size-exclusion chromatography of globular proteins of known Stokes radii ‘Rs’ (see the Materials and Methods section). The arrow indicates the elution position of the N-Oct-3 POU domain.
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Figure 1: Characterization of the N-Oct-3 POU domain. (A) Detection of a single band with the expected N-Oct-3 DBD molecular mass by Coomassie-blue staining in 13% SDS-PAGE (see the molecular mass markers on the right). (B) Dynamic light scattering of the N-Oct-3 DBD (see text). (C) Calibration curve obtained by FPLC size-exclusion chromatography of globular proteins of known Stokes radii ‘Rs’ (see the Materials and Methods section). The arrow indicates the elution position of the N-Oct-3 POU domain.

Mentions: The N-Oct-3 DNA-binding domain (DBD) purifies as a single species of 20 kDa molecular mass as judged by SDS-PAGE (Figure 1A). In order to investigate the oligomerization state and hydrodynamic radius of this POU domain, we first carried out dynamic light scattering (DLS) and analytical gel filtration experiments. DLS measurements recorded at 20°C and at a maximal concentration of 4 mg/ml indicated a low polydispersity and a narrow particle size distribution diagram corresponding to a hydrodynamic radius of 29.3 Å (Figure 1B). The purified N-Oct-3 POU domain eluted from a FPLC-size exclusion chromatography column between the 43 and 25 kDa calibration proteins and the elution volume served to calculate its Stokes radius (Figure 1C). The resulting Rs value of 27.6 Å was very similar to that calculated by DLS, but significantly higher than those of globular proteins of an equivalent molecular weight. This indicates the presence of either a dimer or an elongated monomer in solution.Figure 1.


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)

Characterization of the N-Oct-3 POU domain. (A) Detection of a single band with the expected N-Oct-3 DBD molecular mass by Coomassie-blue staining in 13% SDS-PAGE (see the molecular mass markers on the right). (B) Dynamic light scattering of the N-Oct-3 DBD (see text). (C) Calibration curve obtained by FPLC size-exclusion chromatography of globular proteins of known Stokes radii ‘Rs’ (see the Materials and Methods section). The arrow indicates the elution position of the N-Oct-3 POU domain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Characterization of the N-Oct-3 POU domain. (A) Detection of a single band with the expected N-Oct-3 DBD molecular mass by Coomassie-blue staining in 13% SDS-PAGE (see the molecular mass markers on the right). (B) Dynamic light scattering of the N-Oct-3 DBD (see text). (C) Calibration curve obtained by FPLC size-exclusion chromatography of globular proteins of known Stokes radii ‘Rs’ (see the Materials and Methods section). The arrow indicates the elution position of the N-Oct-3 POU domain.
Mentions: The N-Oct-3 DNA-binding domain (DBD) purifies as a single species of 20 kDa molecular mass as judged by SDS-PAGE (Figure 1A). In order to investigate the oligomerization state and hydrodynamic radius of this POU domain, we first carried out dynamic light scattering (DLS) and analytical gel filtration experiments. DLS measurements recorded at 20°C and at a maximal concentration of 4 mg/ml indicated a low polydispersity and a narrow particle size distribution diagram corresponding to a hydrodynamic radius of 29.3 Å (Figure 1B). The purified N-Oct-3 POU domain eluted from a FPLC-size exclusion chromatography column between the 43 and 25 kDa calibration proteins and the elution volume served to calculate its Stokes radius (Figure 1C). The resulting Rs value of 27.6 Å was very similar to that calculated by DLS, but significantly higher than those of globular proteins of an equivalent molecular weight. This indicates the presence of either a dimer or an elongated monomer in solution.Figure 1.

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