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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 the tandem DNA site DBS. Experiments were performed in 200 mM sodium phosphate (pH 7) and 0.2 mM DTT at 298 K. Each panel shows the ITC data in both raw and integrated, concentration-normalized form. The top panels show direct ITC titrations, i.e., injecting DBS into a cell containing E2C: (A) E2C-16, (C) E2C-11, and (E) E2C-18. The bottom panels show reverse ITC titrations, i.e., injecting E2C into a cell containing DBS: (B) E2C-16, (D) E2C-11, and (F) E2C-18. Lines are global fits (62) to the two-site binding model of direct and reverse titrations for each homologous protein.
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fig3: Binding of E2C to the tandem DNA site DBS. Experiments were performed in 200 mM sodium phosphate (pH 7) and 0.2 mM DTT at 298 K. Each panel shows the ITC data in both raw and integrated, concentration-normalized form. The top panels show direct ITC titrations, i.e., injecting DBS into a cell containing E2C: (A) E2C-16, (C) E2C-11, and (E) E2C-18. The bottom panels show reverse ITC titrations, i.e., injecting E2C into a cell containing DBS: (B) E2C-16, (D) E2C-11, and (F) E2C-18. Lines are global fits (62) to the two-site binding model of direct and reverse titrations for each homologous protein.

Mentions: Binding of E2C-16 to the tandem DBS was investigated by ITC (Figure 1C). Figure 3A shows the experiment conducted by direct titration, i.e., injecting DNA into a cell containing the protein. In the first injections, the large excess of protein leads to predominant formation of the ternary E2C−DBS−E2C complex. The apparent DNA:protein stoichiometry, Ndirect, is on the order of 1:2, and the apparent enthalpy of binding (Figure 3A, approximately −38 kcal/mol) is close to the sum of the ΔH values for binding to isolated BS1 and BS2 (see Table 1). Figure 3B shows the experiment conducted by reverse titration, i.e., injecting protein into a cell containing DNA. As expected for a multisite system, the observed shape of the isotherms depends on the order of addition in the titration experiment (see Figure S2 of the Supporting Information for an explicative scheme). In the first injections, the large excess of DNA leads to predominant formation of the binary E2C−DBS1 and E2C−DBS2 complexes. The apparent protein:DNA stoichiometry Nreverse is on the order of 2:1, and the apparent enthalpy of binding is close to the average of the ΔH values for binding to isolated BS1 and BS2 (compare the ΔH of −20 kcal/mol from Figure 3B with data from Table 1).


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 the tandem DNA site DBS. Experiments were performed in 200 mM sodium phosphate (pH 7) and 0.2 mM DTT at 298 K. Each panel shows the ITC data in both raw and integrated, concentration-normalized form. The top panels show direct ITC titrations, i.e., injecting DBS into a cell containing E2C: (A) E2C-16, (C) E2C-11, and (E) E2C-18. The bottom panels show reverse ITC titrations, i.e., injecting E2C into a cell containing DBS: (B) E2C-16, (D) E2C-11, and (F) E2C-18. Lines are global fits (62) to the two-site binding model of direct and reverse titrations for each homologous protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3091369&req=5

fig3: Binding of E2C to the tandem DNA site DBS. Experiments were performed in 200 mM sodium phosphate (pH 7) and 0.2 mM DTT at 298 K. Each panel shows the ITC data in both raw and integrated, concentration-normalized form. The top panels show direct ITC titrations, i.e., injecting DBS into a cell containing E2C: (A) E2C-16, (C) E2C-11, and (E) E2C-18. The bottom panels show reverse ITC titrations, i.e., injecting E2C into a cell containing DBS: (B) E2C-16, (D) E2C-11, and (F) E2C-18. Lines are global fits (62) to the two-site binding model of direct and reverse titrations for each homologous protein.
Mentions: Binding of E2C-16 to the tandem DBS was investigated by ITC (Figure 1C). Figure 3A shows the experiment conducted by direct titration, i.e., injecting DNA into a cell containing the protein. In the first injections, the large excess of protein leads to predominant formation of the ternary E2C−DBS−E2C complex. The apparent DNA:protein stoichiometry, Ndirect, is on the order of 1:2, and the apparent enthalpy of binding (Figure 3A, approximately −38 kcal/mol) is close to the sum of the ΔH values for binding to isolated BS1 and BS2 (see Table 1). Figure 3B shows the experiment conducted by reverse titration, i.e., injecting protein into a cell containing DNA. As expected for a multisite system, the observed shape of the isotherms depends on the order of addition in the titration experiment (see Figure S2 of the Supporting Information for an explicative scheme). In the first injections, the large excess of DNA leads to predominant formation of the binary E2C−DBS1 and E2C−DBS2 complexes. The apparent protein:DNA stoichiometry Nreverse is on the order of 2:1, and the apparent enthalpy of binding is close to the average of the ΔH values for binding to isolated BS1 and BS2 (compare the ΔH of −20 kcal/mol from Figure 3B with data from Table 1).

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