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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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Conformational search by torsion driving. (A) Location of the linker (brown-coded) within the sequence of the N-Oct-3 DBD: the Gly 98 and Gly 110 residues (highlighted) flank the SPTSIDKIAAQ undecapeptide (underlined). Other critical features are the Gln 63 and Asn 162 residues (red-coded) in the respective POUs and POUh DNA recognition helices (purple-coded). Display code for the remaining elements as follows: gray for the POUh N-terminal arm, blue for helices 1, 2, 4, 5, 6, green for the regions between secondary structure elements, black for exogenous regions resulting from the DBD cloning. (B–D) Clustering of molecular mechanics-derived structures in families of potential free forms (B, C) and extended conformers (D). The conformers Cα traces are structurally aligned within a 4–5 Å R.M.S. range in each cluster. (E–G) The conformers Cf 183 (E), Cf 194 (F) and Cf 221 (G) are the best representatives of each family, respectively FI (B), FII (C) and NF (D). In all cases, Gly 98 and Gly 110 are coded in brown, Gln 63 and Asn 162 in red, the POUs and POUh recognition helices in purple. RHdist is monitored in Å.
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Figure 6: Conformational search by torsion driving. (A) Location of the linker (brown-coded) within the sequence of the N-Oct-3 DBD: the Gly 98 and Gly 110 residues (highlighted) flank the SPTSIDKIAAQ undecapeptide (underlined). Other critical features are the Gln 63 and Asn 162 residues (red-coded) in the respective POUs and POUh DNA recognition helices (purple-coded). Display code for the remaining elements as follows: gray for the POUh N-terminal arm, blue for helices 1, 2, 4, 5, 6, green for the regions between secondary structure elements, black for exogenous regions resulting from the DBD cloning. (B–D) Clustering of molecular mechanics-derived structures in families of potential free forms (B, C) and extended conformers (D). The conformers Cα traces are structurally aligned within a 4–5 Å R.M.S. range in each cluster. (E–G) The conformers Cf 183 (E), Cf 194 (F) and Cf 221 (G) are the best representatives of each family, respectively FI (B), FII (C) and NF (D). In all cases, Gly 98 and Gly 110 are coded in brown, Gln 63 and Asn 162 in red, the POUs and POUh recognition helices in purple. RHdist is monitored in Å.

Mentions: Before dealing with the structural determinants of N-Oct-3 linker flexibility, we first need to recall its distinctive features. Using circular dichroism, we previously observed an increase in the α-helical content of the N-Oct-3 DBD when binding to its DNA targets, in contrast to the Oct-1 DBD (38). Since the only significant difference between these two highly conserved DBDs is their respective linker sequences, we engineered chimeric proteins where the N-Oct-3 and the Oct-1 linkers were interchanged. This showed that the replacement of the N-Oct-3 DBD linker by that of Oct-1 abolished the increase in α-helical structure, whereas the replacement of the Oct-1 linker by that of N-Oct-3 resulted in the typical increase in the α-helical content following protein/DNA complex formation. Since a number of reliable secondary structure prediction methods indicated that the heptapeptide motif IDKIAAQ specific to the N-Oct-3 linker could adopt an α-helical structure, we built another set of chimeric proteins where this heptapeptide was removed from the N-Oct-3 linker and embedded within the Oct-1 linker. As the results were similar to those for the entire linker interchange experiments, we concluded that the ability of the N-Oct-3 linker to adopt an α-helical structure when binding to a DNA target could be ascribed to the IDKIAAQ motif (see its location in the DBD sequence in Figure 6A). We now show that the potential secondary structure of this heptapeptide motif can also be stabilized independently of DNA binding, when free DBD concentrations are greater than 0.7 mg/ml (see Figure S1 and its legend), which are the conditions of the hydrodynamic and SAXS experiments reported here. Note that the link between protein folding and molecular concentration has been revealed in a number of recent works [see for example (39,40)]. Thus the N-Oct-3 linker has the characteristics of a ‘helical linker’ as defined by George and Heringa based on an extensive compilation of inter-domain linkers (41). Interestingly, the helical heptapeptide IDKIAAQ is preceded by the 4-residue motif SPTS (Figure 6A), shown to form a β-turn in a number of proteins and polypeptides, the structures of which were solved by crystallography or NMR (42–44).Figure 6.


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)

Conformational search by torsion driving. (A) Location of the linker (brown-coded) within the sequence of the N-Oct-3 DBD: the Gly 98 and Gly 110 residues (highlighted) flank the SPTSIDKIAAQ undecapeptide (underlined). Other critical features are the Gln 63 and Asn 162 residues (red-coded) in the respective POUs and POUh DNA recognition helices (purple-coded). Display code for the remaining elements as follows: gray for the POUh N-terminal arm, blue for helices 1, 2, 4, 5, 6, green for the regions between secondary structure elements, black for exogenous regions resulting from the DBD cloning. (B–D) Clustering of molecular mechanics-derived structures in families of potential free forms (B, C) and extended conformers (D). The conformers Cα traces are structurally aligned within a 4–5 Å R.M.S. range in each cluster. (E–G) The conformers Cf 183 (E), Cf 194 (F) and Cf 221 (G) are the best representatives of each family, respectively FI (B), FII (C) and NF (D). In all cases, Gly 98 and Gly 110 are coded in brown, Gln 63 and Asn 162 in red, the POUs and POUh recognition helices in purple. RHdist is monitored in Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Conformational search by torsion driving. (A) Location of the linker (brown-coded) within the sequence of the N-Oct-3 DBD: the Gly 98 and Gly 110 residues (highlighted) flank the SPTSIDKIAAQ undecapeptide (underlined). Other critical features are the Gln 63 and Asn 162 residues (red-coded) in the respective POUs and POUh DNA recognition helices (purple-coded). Display code for the remaining elements as follows: gray for the POUh N-terminal arm, blue for helices 1, 2, 4, 5, 6, green for the regions between secondary structure elements, black for exogenous regions resulting from the DBD cloning. (B–D) Clustering of molecular mechanics-derived structures in families of potential free forms (B, C) and extended conformers (D). The conformers Cα traces are structurally aligned within a 4–5 Å R.M.S. range in each cluster. (E–G) The conformers Cf 183 (E), Cf 194 (F) and Cf 221 (G) are the best representatives of each family, respectively FI (B), FII (C) and NF (D). In all cases, Gly 98 and Gly 110 are coded in brown, Gln 63 and Asn 162 in red, the POUs and POUh recognition helices in purple. RHdist is monitored in Å.
Mentions: Before dealing with the structural determinants of N-Oct-3 linker flexibility, we first need to recall its distinctive features. Using circular dichroism, we previously observed an increase in the α-helical content of the N-Oct-3 DBD when binding to its DNA targets, in contrast to the Oct-1 DBD (38). Since the only significant difference between these two highly conserved DBDs is their respective linker sequences, we engineered chimeric proteins where the N-Oct-3 and the Oct-1 linkers were interchanged. This showed that the replacement of the N-Oct-3 DBD linker by that of Oct-1 abolished the increase in α-helical structure, whereas the replacement of the Oct-1 linker by that of N-Oct-3 resulted in the typical increase in the α-helical content following protein/DNA complex formation. Since a number of reliable secondary structure prediction methods indicated that the heptapeptide motif IDKIAAQ specific to the N-Oct-3 linker could adopt an α-helical structure, we built another set of chimeric proteins where this heptapeptide was removed from the N-Oct-3 linker and embedded within the Oct-1 linker. As the results were similar to those for the entire linker interchange experiments, we concluded that the ability of the N-Oct-3 linker to adopt an α-helical structure when binding to a DNA target could be ascribed to the IDKIAAQ motif (see its location in the DBD sequence in Figure 6A). We now show that the potential secondary structure of this heptapeptide motif can also be stabilized independently of DNA binding, when free DBD concentrations are greater than 0.7 mg/ml (see Figure S1 and its legend), which are the conditions of the hydrodynamic and SAXS experiments reported here. Note that the link between protein folding and molecular concentration has been revealed in a number of recent works [see for example (39,40)]. Thus the N-Oct-3 linker has the characteristics of a ‘helical linker’ as defined by George and Heringa based on an extensive compilation of inter-domain linkers (41). Interestingly, the helical heptapeptide IDKIAAQ is preceded by the 4-residue motif SPTS (Figure 6A), shown to form a β-turn in a number of proteins and polypeptides, the structures of which were solved by crystallography or NMR (42–44).Figure 6.

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
Related in: MedlinePlus