HMGB binding to DNA: single and double box motifs

Abstract

High mobility group (HMG) proteins are nuclear proteins believed to significantly affect DNA interactions by altering nucleic acid flexibility. Group B (HMGB) proteins contain HMG box domains known to bind to the DNA minor groove without sequence specificity, slightly intercalating base pairs and inducing a strong bend in the DNA helical axis. A dual-beam optical tweezers system is used to extend double-stranded DNA (dsDNA) in the absence as well as presence of a single box derivative of human HMGB2 [HMGB2(box A)] and a double box derivative of rat HMGB1 [HMGB1(box A+box B)]. The single box domain is observed to reduce the persistence length of the double helix, generating sharp DNA bends with an average bending angle of 99+/-9 degrees and, at very high concentrations, stabilizing dsDNA against denaturation. The double box protein contains two consecutive HMG box domains joined by a flexible tether. This protein also reduces the DNA persistence length, induces an average bending angle of 77+/-7 degrees , and stabilizes dsDNA at significantly lower concentrations. These results suggest that single and double box proteins increase DNA flexibility and stability, albeit both effects are achieved at much lower protein concentrations for the double box. In addition, at low concentrations, the single box protein can alter DNA flexibility without stabilizing dsDNA, whereas stabilization at higher concentrations is likely achieved through a cooperative binding mode.

Figure 1

Structures of HMGB proteins bound to dsDNA. a) Single HMG box (purple) bound to dsDNA (green and yellow) in this crystal structure for HMGB family member HMG–D protein from Drosophila melanogaster. The non-sequence specific binding is characterized by van der Waals contacts, direct and water-mediated hydrogen bonding and partial intercalation, yielding a strong, continuous deformation of the DNA backbone. b) Structure of tandem HMG boxes from an artificial HMGB protein created with transcription factor SRY in place of box A of HMGB1. This artificial tandem SRY/B box protein (red/blue) binds to a 16-bp sequence of double-stranded DNA with each HMG box engaging DNA similarly, though the SRY box is sequence specific (allowing determination of this NMR structure). The domains are connected by a basic sequence that is believed to be flexible. c) Rotated view of the structure from (b) revealing that each HMG box domain induces an independent DNA bend, with the bends partially reinforcing. The plane of the first and last base pairs in (a) and (c) are perpendicular to the paper. Dotted green arrows indicate the direction of the helical axis, and the deflection of the outgoing arrows give the protein induced bending angle. Images were generated using coordinates from the NCBI Protein Data Bank (entries 1QRV and 2GZK) with Swiss View/DeepView v3.7 software.

Figure 2

HMGB proteins lower the persistence length of dsDNA. a) Averaged stretching (solid) and relaxation (open) data for dsDNA in the absence (blue circles) or presence (green boxes) of 3 nM HMGB1(box A+B) in 10 mM Hepes (pH 7.5) and 100 nM Na⁺. Solid and dotted lines are averages for the extension and relaxation data, respectively. Each line is the average of four data sets, collected at 100 nm intervals and each point averaged for 1.0 second. b) Averaged stretching/relaxation data shown separately, revealing minimal hysteresis. Standard uncertainty in each point is 1 pN or less, within the size of each symbol. c) Levenberg – Marquardt fits (solid lines) to the force extension data according to the Worm-Like Chain (WLC) model, as described in the text. The fits yielded b_ds = 0.339 ± 0.001 nm/bp, K_ds = 1200 ± 50 pN and P_ds = 46 ± 2 nm for dsDNA, and b_ds = 0.339 ± 0.003 nm/bp, K_ds = 939 ± 100 pN and P_ds = 22 ± 2 nm for dsDNA with 3 nM HMGB1(box A+B). Fits are limited to data below 45 pN.

Figure 3

Binding isotherms for the change in DNA persistence length (P_ds) as a function of HMGB protein concentration (c). Each data point corresponds to the average of 3 to 4 extension curves, as shown in Figure 2, which are fit to the WLC model to obtain P_ds. With increasing protein concentration, both the single box HMGB2(box A) (blue circles) and the double box HMGB1(box A+B) (green boxes) proteins substantially reduce the persistence length of dsDNA. The double box HMGB1(box A+B) protein shows a stronger equilibrium association constant per ligand (K). The solid lines are fits to the binding isotherms described in the text, yielding K = 0.11 ± 0.05 × 10⁹ M⁻¹ for HMGB2(box A) and K = 11 ± 3 × 10⁹ M⁻¹ for HMGB1(box A+B).

Figure 4

HMGB1(boxes A+B) protein stabilizes the DNA melting transition. a) Full stretching (solid) and relaxation (dotted) cycles for HMGB1(boxes A+B) bound to DNA. In addition to the change in the persistence length discussed above, the melting transition is stabilized by the addition of 2 nM (blue), 4 nM (green), 10 nM (gold) and 100 nM (red) of protein. Furthermore, there is an increase in the slope of the melting transition and an increase in the hysteresis evident upon relaxation. b) Enlargement of the melting transition shown in a). c) Stretching and relaxation cycles for the single box HMGB2(box A) show little change from bare DNA (black) through 2 nM (blue) and 4 nM (green) of protein. At and above 10 nm of protein concentration (gold and red), DNA stabilization becomes evident, due to cooperative binding to dsDNA. d) Enlargement of the melting transition shown in c).

Figure 5

Binding isotherms for the change in DNA melting force (ΔF_m) as a function of concentrations (c) of added HMGB protein and ions. Solid data points and lines are for DNA/protein complexes in 100 mM Na⁺, while open data points and dotted lines are for binding in 50 mM Na⁺. Each point represents an average over 4 – 10 extension cycles. a) Over relatively low concentrations (< 10 nM protein), there is little change in the melting force for dsDNA bound to HMGB2(box A), while HMGB1(box A+B) strongly stabilizes the nucleic acid. b) At higher concentrations, stabilization is evident for the single box domain, due to cooperative binding to DNA. While the cooperative binding of HMGB2(box A) appears to be unaffected by salt concentration over this range, the binding of HMGB1(box A+B) is strongly enhanced in low salt, due possibly to an increase in cooperativity. In 100 mM Na⁺ the fits to binding models determine K = 0.028 ± 0.010 × 10⁹ M⁻¹ for HMGB2(box A) and K = 13 ± 3 × 10⁹ M⁻¹ for HMGB1(box A+B), while in 50 mM Na⁺, K = 0.014 ± 0.006× 10⁹ M⁻¹ for HMGB2(box A) and K = 7 ± 2 × 10⁹ M⁻¹ for HMGB1(box A+B).

Figure 6

Stretching curves for dsDNA. a) Force extension (filled blue) and relaxation (open blue) curves for double stranded dsDNA in 10 mM Hepes (pH 7.5) including 100 nM Na⁺. Low forces reveal an entropic spring regime, while the extension at higher forces is described enthalpically, as the chain stretches and unwinds. At ~ 62 pN, dsDNA abruptly lengthens, as bases pairs cooperatively melt and base stacking is disrupted. Relaxing the nucleic acid allows reannealing, while continuing to higher forces extends the predominantly single stranded construct, until strand separation is reached (green and red). Solid lines are models of polymer elasticity described in the text, and the area between them determines the free energy of melting. b) Trapping multiple strands of labeled dsDNA reveals melting plateaus at integer multiples of the dsDNA melting force, and confirms the linearity of the force measurement. This dual beam instrument can examine trapping forces up to ~ 325 pN.