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Thermodynamics of cooperative DNA recognition at a replication origin and transcription regulatory site.

Dellarole M, Sánchez IE, de Prat Gay G - Biochemistry (2010)

Bottom Line: This cooperativity is associated with a change in DNA structure, where the overall B conformation is maintained.Because the DNA binding helix is almost identical in the three domains, the differences must lie dispersed throughout this unique dimeric β-barrel fold.This is in surprising agreement with previous results for this domain, which revealed a strong coupling between global dynamics and DNA recognition.

View Article: PubMed Central - PubMed

Affiliation: Protein Structure-Function and Engineering Laboratory, Fundación Instituto Leloir and IIBBA-Conicet, Patricias Argentinas 435, Buenos Aires, Argentina.

ABSTRACT
Binding cooperativity guides the formation of protein-nucleic acid complexes, in particular those that are highly regulated such as replication origins and transcription sites. Using the DNA binding domain of the origin binding and transcriptional regulator protein E2 from human papillomavirus type 16 as model, and through isothermal titration calorimetry analysis, we determined a positive, entropy-driven cooperativity upon binding of the protein to its cognate tandem double E2 site. This cooperativity is associated with a change in DNA structure, where the overall B conformation is maintained. Two homologous E2 domains, those of HPV18 and HPV11, showed that the enthalpic-entropic components of the reaction and DNA deformation can diverge. Because the DNA binding helix is almost identical in the three domains, the differences must lie dispersed throughout this unique dimeric β-barrel fold. This is in surprising agreement with previous results for this domain, which revealed a strong coupling between global dynamics and DNA recognition.

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Binding of E2C to BS1 and BS2. Experiments were performed in 200 mM sodium phosphate (pH 7) and 0.2 mM DTT at 298 K. The top panels show integrated, concentration-normalized binding isotherms of BS1 (blue) or BS2 (red) injected into the ITC cell containing protein. The solid lines represent global fits (62) to a single-site binding model of data points of at least two independent experiments for each homologous protein: (A) E2C-16, (D) E2C-11, and (G) E2C-18. The middle panels show difference CD spectra for binding of E2C to BS1 (blue) and BS2 (red): (B) E2C-16, (E) E2C-11, and (H) E2C-18. The spectra of E2C and BS1 or BS2 were subtracted from the spectra of each 1:1 E2C complex. All data are shown to the same scale for direct comparison. The bottom panels show difference absorbance spectra for binding of E2C to BS1 (blue) and BS2 (red): (C) E2C-16, (F) E2C-11, and (I) E2C-18. The spectra of E2C and BS1 or BS2 were subtracted from the spectra of each 1:1 E2C complex.
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fig2: Binding of E2C to BS1 and BS2. Experiments were performed in 200 mM sodium phosphate (pH 7) and 0.2 mM DTT at 298 K. The top panels show integrated, concentration-normalized binding isotherms of BS1 (blue) or BS2 (red) injected into the ITC cell containing protein. The solid lines represent global fits (62) to a single-site binding model of data points of at least two independent experiments for each homologous protein: (A) E2C-16, (D) E2C-11, and (G) E2C-18. The middle panels show difference CD spectra for binding of E2C to BS1 (blue) and BS2 (red): (B) E2C-16, (E) E2C-11, and (H) E2C-18. The spectra of E2C and BS1 or BS2 were subtracted from the spectra of each 1:1 E2C complex. All data are shown to the same scale for direct comparison. The bottom panels show difference absorbance spectra for binding of E2C to BS1 (blue) and BS2 (red): (C) E2C-16, (F) E2C-11, and (I) E2C-18. The spectra of E2C and BS1 or BS2 were subtracted from the spectra of each 1:1 E2C complex.

Mentions: We conducted the analysis of the cognate interaction between E2C-16 and DBS (Figure 1D). Throughout this work, all experiments were performed in 200 mM sodium phosphate (pH 7) and 0.2 mM DTT at 298 K. To determine all binding constants in Scheme 1 with reliability, we first investigated binding of E2C-16 to the isolated sites BS1 and BS2 (Figure 1D) by ITC. Figure 2 shows integrated, concentration-normalized data for injection of BS1 (blue circles) or BS2 (red circles) into the cell containing E2C-16 (Figure 2A). The observed reaction stoichiometry is ≥0.8, and the c value is >10 (Table 1), validating fitting a 1:1 binding model to the data. The fit yields the binding free energies (ΔG) and their enthalpic and entropic components (Table 1). Binding of E2C-16 to the individual cognate sites was enthalpy-driven in both cases, where binding to BS2 was significantly stronger than binding to BS1 (4-fold difference in the equilibrium binding constant, K).


Thermodynamics of cooperative DNA recognition at a replication origin and transcription regulatory site.

Dellarole M, Sánchez IE, de Prat Gay G - Biochemistry (2010)

Binding of E2C to BS1 and BS2. Experiments were performed in 200 mM sodium phosphate (pH 7) and 0.2 mM DTT at 298 K. The top panels show integrated, concentration-normalized binding isotherms of BS1 (blue) or BS2 (red) injected into the ITC cell containing protein. The solid lines represent global fits (62) to a single-site binding model of data points of at least two independent experiments for each homologous protein: (A) E2C-16, (D) E2C-11, and (G) E2C-18. The middle panels show difference CD spectra for binding of E2C to BS1 (blue) and BS2 (red): (B) E2C-16, (E) E2C-11, and (H) E2C-18. The spectra of E2C and BS1 or BS2 were subtracted from the spectra of each 1:1 E2C complex. All data are shown to the same scale for direct comparison. The bottom panels show difference absorbance spectra for binding of E2C to BS1 (blue) and BS2 (red): (C) E2C-16, (F) E2C-11, and (I) E2C-18. The spectra of E2C and BS1 or BS2 were subtracted from the spectra of each 1:1 E2C complex.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3091369&req=5

fig2: Binding of E2C to BS1 and BS2. Experiments were performed in 200 mM sodium phosphate (pH 7) and 0.2 mM DTT at 298 K. The top panels show integrated, concentration-normalized binding isotherms of BS1 (blue) or BS2 (red) injected into the ITC cell containing protein. The solid lines represent global fits (62) to a single-site binding model of data points of at least two independent experiments for each homologous protein: (A) E2C-16, (D) E2C-11, and (G) E2C-18. The middle panels show difference CD spectra for binding of E2C to BS1 (blue) and BS2 (red): (B) E2C-16, (E) E2C-11, and (H) E2C-18. The spectra of E2C and BS1 or BS2 were subtracted from the spectra of each 1:1 E2C complex. All data are shown to the same scale for direct comparison. The bottom panels show difference absorbance spectra for binding of E2C to BS1 (blue) and BS2 (red): (C) E2C-16, (F) E2C-11, and (I) E2C-18. The spectra of E2C and BS1 or BS2 were subtracted from the spectra of each 1:1 E2C complex.
Mentions: We conducted the analysis of the cognate interaction between E2C-16 and DBS (Figure 1D). Throughout this work, all experiments were performed in 200 mM sodium phosphate (pH 7) and 0.2 mM DTT at 298 K. To determine all binding constants in Scheme 1 with reliability, we first investigated binding of E2C-16 to the isolated sites BS1 and BS2 (Figure 1D) by ITC. Figure 2 shows integrated, concentration-normalized data for injection of BS1 (blue circles) or BS2 (red circles) into the cell containing E2C-16 (Figure 2A). The observed reaction stoichiometry is ≥0.8, and the c value is >10 (Table 1), validating fitting a 1:1 binding model to the data. The fit yields the binding free energies (ΔG) and their enthalpic and entropic components (Table 1). Binding of E2C-16 to the individual cognate sites was enthalpy-driven in both cases, where binding to BS2 was significantly stronger than binding to BS1 (4-fold difference in the equilibrium binding constant, K).

Bottom Line: This cooperativity is associated with a change in DNA structure, where the overall B conformation is maintained.Because the DNA binding helix is almost identical in the three domains, the differences must lie dispersed throughout this unique dimeric β-barrel fold.This is in surprising agreement with previous results for this domain, which revealed a strong coupling between global dynamics and DNA recognition.

View Article: PubMed Central - PubMed

Affiliation: Protein Structure-Function and Engineering Laboratory, Fundación Instituto Leloir and IIBBA-Conicet, Patricias Argentinas 435, Buenos Aires, Argentina.

ABSTRACT
Binding cooperativity guides the formation of protein-nucleic acid complexes, in particular those that are highly regulated such as replication origins and transcription sites. Using the DNA binding domain of the origin binding and transcriptional regulator protein E2 from human papillomavirus type 16 as model, and through isothermal titration calorimetry analysis, we determined a positive, entropy-driven cooperativity upon binding of the protein to its cognate tandem double E2 site. This cooperativity is associated with a change in DNA structure, where the overall B conformation is maintained. Two homologous E2 domains, those of HPV18 and HPV11, showed that the enthalpic-entropic components of the reaction and DNA deformation can diverge. Because the DNA binding helix is almost identical in the three domains, the differences must lie dispersed throughout this unique dimeric β-barrel fold. This is in surprising agreement with previous results for this domain, which revealed a strong coupling between global dynamics and DNA recognition.

Show MeSH
Related in: MedlinePlus