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

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.

Figure 1

E2 binding sites in the alpha papillomavirus upstream regulatory region. (A) Schematic view of the upstream regulatory region of a prototypical alpha papillomavirus genome. Shown are flanking ORFs L1 and E6, E2 binding sites BS1, BS2, BS3, and BS4, the E1 binding site, the Sp1 binding site, and the TATA box. (B) Sequence logo (57,58) of the cis-responsive elements of the E6 promoter. (C) Model of the complex of two E2C homodimers with two adjacent sites separated by one base. Under our experimental conditions, E2C is always dimeric. The model was constructed by aligning two copies of a HPV6 E2C−DNA complex (PDB entry 2ayg) using PyMol (Delano Scientific LLC). (D) Sequence of the double-stranded oligonucleotide DBS from HPV16, with BS1 colored blue and BS2 red.

Scheme 1

Figure 2

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.

Figure 3

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.

Figure 4

Cooperative binding of E2C to the tandem site DBS. The top panels show thermodynamic dissection of (A) E2C-16, (C) E2C-11, or (E) E2C-18 binding to DBS. Negative values represent a favorable additional contribution to binding, whereas positive values represent an unfavorable additional contribution to binding. The bottom panels show the fractional population of free DBS, DBS with one site occupied, and DBS with both sites occupied as a function of (B) E2C-16, (D) E2C-11, or (F) E2C-18 concentration. Solid lines were calculated using the measured values of K_BS1, K_BS2, and K_coop. Dotted lines were calculated using the measured values of K_BS1, K_BS2, and K_coop set to 1.

Figure 5

Spectroscopic changes associated with 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. Solid lines are difference spectra for binding of E2C to DBS. The spectra of DBS and two E2C molecules were subtracted from the spectra of the E2C−DBS−E2C ternary complex. Dashed lines are sums of difference spectra for the E2C−BS1 and E2C−BS2 complexes. The top panels show difference CD spectra: (A) E2C-16, (C) E2C-11, and (E) E2C-18. The bottom panels show difference absorbance spectra: (B) E2C-16, (D) E2C-11, and (F) E2C-18.

Figure 6

Kinetics of structural changes upon binding of E2C to the tandem DNA site DBS. DBS (1 μM) was mixed with 2 μM E2C-11 in 200 mM sodium phosphate (pH 7) and 1 mM DTT at 298 K, and the changes in absorbance at 270 nm were followed. Shown are the normalized data and the fit to a single-exponential function.