The long acidic tail of high mobility group box 1 (HMGB1) protein forms an extended and flexible structure that interacts with specific residues within and between the HMG boxes

HMGB1 (high mobility group B1) is a conserved chromosomal protein composed of two similar DNA binding domains (HMG box A and box B) linked by a short basic stretch to an acidic C-terminal tail of 30 residues. The acidic tail modulates the DNA binding properties of HMGB1, and its length differentiates the various HMGB family members. We synthesized a peptide that corresponds to the acidic tail in HMGB1 (T-peptide) and studied its binding to the single boxes and to the fragment corresponding to tailless HMGB1 (designated as AB(bt) fragment). CD spectroscopy showed that T-peptide stabilizes significantly the AB(bt) fragment and that the complex has an identical thermal stability as full-length HMGB1. Calorimetric and NMR data showed that T-peptide binds with a dissociation constant of 9 microM to box A and much more weakly to box B. (1)H-(15)N HSQC spectra of full-length HMGB1 and of the AB(bt) fragment are very similar; the small chemical shift differences that exist correspond to those residues of the AB(bt) fragment that were affected by the addition of the T-peptide. We conclude that the T-peptide mimics closely the acidic tail and that the basic stretch and the acidic tail form an extended and flexible segment. The tail interacts with specific residues in the boxes and shields them from other interactions.     

A critical role in structure-specific DNA binding for the acetylatable lysine residues in HMGB1

The structure-specific DNA-binding protein HMGB1 (high-mobility group protein B1) which comprises two tandem HMG boxes (A and B) and an acidic C-terminal tail, is acetylated in vivo at Lys(2) and Lys(11) in the A box. Mutation to alanine of both residues in the isolated A domain, which has a strong preference for pre-bent DNA, abolishes binding to four-way junctions and 88 bp DNA minicircles. The same mutations in full-length HMGB1 also abolish its binding to four-way junctions, and binding to minicircles is substantially impaired. In contrast, when the acidic tail is absent (AB di-domain) there is little effect of the double mutation on four-way junction binding, although binding to minicircles is reduced approximately 15-fold. Therefore it appears that in AB the B domain is able to substitute for the non-functional A domain, whereas in full-length HMGB1 the B domain is masked by the acidic tail. In no case does single substitution of Lys(2) or Lys(11) abolish DNA binding. The double mutation does not significantly perturb the structure of the A domain. We conclude that Lys(2) and Lys(11) are critical for binding of the isolated A domain and HMGB1 to distorted DNA substrates.     

Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein

Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain. The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect. Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1. To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used. These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain. The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions. In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA. Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.     

Interactions between N- and C-terminal domains of the Saccharomyces cerevisiae high-mobility group protein HMO1 are required for DNA bending

The Saccharomyces cerevisiae high-mobility group protein HMO1 is composed of two DNA-binding domains termed box A and box B, of which only box B is predicted to adopt a HMG fold, and a lysine-rich C-terminal extension. To assess the interaction between individual domains and their contribution to DNA binding, several HMO1 variants were analyzed. Using circular dichroism spectroscopy, thermal stability was measured. While the melting temperatures of HMO1-boxA and HMO1-boxB are 57.2 and 47.2 degrees C, respectively, HMO1-boxBC, containing box B and the entire C-terminal tail, melts at 46.1 degrees C, suggesting little interaction between box B and the tail. In contrast, full-length HMO1 exhibits a single melting transition at 47.9 degrees C, indicating that interaction between box A and either box B or the tail destabilizes this domain. As HMO1-boxAB, lacking only the lysine-rich C-terminal segment, exhibits two melting transitions at 46.0 and 63.3 degrees C, we conclude that the destabilization of the box A domain seen in full-length HMO1 is due primarily to its interaction with the lysine-rich tail. Determination of DNA substrate specificity using electrophoretic mobility shift assays shows unexpectedly that the lysine-rich tail does not increase DNA binding affinity but instead is required for DNA bending by full-length HMO1; HMO1-boxBC, lacking the box A domain, also fails to bend DNA. In contrast, both HMO1 and HMO1-boxAB, but not the individual HMG domains, exhibit preferred binding to constrained DNA minicircles. Taken together, our data suggest that interactions between box A and the C-terminal tail induce a conformation that is required for DNA bending.     

Visualization of DNA complexes with HMGB1 and its C-truncated form HMGB1(A+B)

Circular dichroism and scanning probe microscopy were used to characterize the interaction of DNA with the nonhistone chromatin protein HMGB1 and its recombinant version HMGB1(A+B) devoid of the C-terminal acidic region. The AFM data corroborate the earlier suggestion concerning the action of tandem DNA-binding domains, and support a modulatory role of the C-terminal domain in HMG protein-DNA interactions.     

Tail-Mediated Collapse of HMGB1 Is Dynamic and Occurs via Differential Binding of the Acidic Tail to the A and B Domains

The architectural DNA-binding protein HMGB1 consists of two tandem HMG-box domains joined by a basic linker to a C-terminal acidic tail, which negatively regulates HMGB1-DNA interactions by binding intramolecularly to the DNA-binding faces of both basic HMG boxes. Here we demonstrate, using NMR chemical-shift mapping at different salt concentrations, that the tail has a higher affinity for the B box and that A box-tail interactions are preferentially disrupted. Previously, we proposed a model in which the boxes are brought together in a collapsed, tail-mediated assembly, which is in dynamic equilibrium with a more extended form. Small-angle X-ray scattering data are consistent with such a dynamic equilibrium between collapsed and extended structures and are best represented by an ensemble. The ensembles contain a significantly higher proportion of collapsed structures when the tail is present. ¹⁵N NMR relaxation measurements show that full-length HMGB1 has a significantly lower rate of rotational diffusion than the tail-less protein, consistent with the loss of independent domain motions in an assembled complex. Mapping studies using the paramagnetic spin label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidin-3-yl)methyl methanethiosulfonate] placed at three locations in the tail confirm our previous findings that the tail binds to both boxes with some degree of specificity. The end of the tail lies further from the body of the protein and is therefore potentially free to interact with other proteins. MTSL labelling at a single site in the A domain (C44) causes detectable relaxation enhancements of B domain residues, suggesting the existence of a “sandwich”-like collapsed structure in which the tail enables the close approach of the basic domains. These intramolecular interactions are presumably important for the dynamic association of HMGB1 with chromatin and provide a mechanism by which protein-protein interactions or posttranslational modifications might regulate the function of the protein at particular sites, or at particular stages in the cell cycle.     

Mapping intramolecular interactions between domains in HMGB1 using a tail-truncation approach

The mechanism underlying negative regulation of HMGB1-DNA interaction by the acidic C-terminal tail is ill defined. To address this issue, we have devised a novel NMR chemical-shift perturbation mapping strategy to elucidate interactions between the tail, which consists solely of aspartic acid and glutamic acid residues, and the two well characterized HMG-box DNA-binding domains. A series of HMGB1 tail-truncation mutants differing from each other by five residues was generated. Chemical-shift perturbation mapping using these mutants shows that tails of different lengths bind with different affinities. Nevertheless, the truncated tails bind along the same path on the HMG boxes as the full-length tail, differences in length being manifested in differences in the “reach”. The tail makes extensive contacts with the DNA-binding surfaces of both HMG boxes, thus explaining the basis of negative regulation of HMGB1–DNA interaction by the tail.     

Native HMGB1 protein inhibits repair of cisplatin-damaged nucleosomes in vitro

The high mobility group box (HMGB) 1 protein, one of the most abundant nuclear non-histone proteins has been known for its inhibitory effect on repair of DNA damaged by the antitumor drug cisplatin. Here, we report the first results that link HMGB1 to repair of cisplatin-treated DNA at nucleosome level. Experiments were carried out with three types of reconstituted nucleosomes strongly positioned on the damaged DNA: linker DNA containing nucleosomes (centrally and end-positioned) and core particles. The highest repair synthesis was registered with end-positioned nucleosomes, two and three times more efficient than that with centrally positioned nucleosomes and core particles, respectively. HMGB1 inhibited repair of linker DNA containing nucleosomes more efficiently than that of core particles. Just the opposite was the effect of the in vivo acetylated HMGB1: stronger repair inhibition was obtained with core particles. No inhibition was observed with HMGB1 lacking the acidic tail. Binding of HMGB1 proteins to different nucleosomes was also analysed. HMGB1 bound preferentially to damage nucleosomes containing linker DNA, while the binding of the acetylated protein was linker independent. We show that both the repair of cisplatin-damaged nucleosomes and its inhibition by HMGB1 are nucleosome position-dependent events which are accomplished via the acidic tail and modulated by acetylation.     

Determinants of specific binding of HMGB1 protein to hemicatenated DNA loops

Protein HMGB1 has long been known as one of the most abundant non-histone proteins in the nucleus of mammalian cells, and has regained interest recently for its function as an extracellular cytokine. As a DNA-binding protein, HMGB1 facilitates DNA-protein interactions by increasing the flexibility of the double helix, and binds specifically to distorted DNA structures. We have previously observed that HMGB1 binds with extremely high affinity to a novel DNA structure, hemicatenated DNA loops (hcDNA), in which double-stranded DNA fragments containing a tract of poly(CA).poly(TG) form a loop maintained at its base by a hemicatenane. Here, we show that the single HMGB1 domains A and B, the HMG-box domain of sex determination factor SRY, as well as the prokaryotic HMGB1-like protein HU, specifically interact with hcDNA (Kd approximately 0.5 nM). However, the affinity of full-length HMGB1 for hcDNA is three orders of magnitude higher (Kd<0.5 pM) and requires the simultaneous presence of both HMG-box domains A and B plus the acidic C-terminal tail on the molecule. Interestingly, the high affinity of the full-length protein for hcDNA does not decrease in the presence of magnesium. Experiments including a comparison of HMGB1 binding to hcDNA and to minicircles containing the CA/TG sequence, binding studies with HMGB1 mutated at intercalating amino acid residues (involved in recognition of distorted DNA structures), and exonuclease III footprinting, strongly suggest that the hemicatenane, not the DNA loop, is the main determinant of the affinity of HMGB1 for hcDNA. Experiments with supercoiled CA/TG-minicircles did not reveal any involvement of left-handed Z-DNA in HMGB1 binding. Our results point to a tight structural fit between HMGB1 and DNA hemicatenanes under physiological conditions, and suggest that one of the nuclear functions of HMGB1 could be linked to the possible presence of hemicatenanes in the cell.     

Expression, purification and characterization of TAT-high mobility group box-1A peptide as a carrier of nucleic acids

High mobility group box 1 (HMGB1) is an abundant nuclear protein that binds to double-stranded DNA. HMGB1 is composed of high mobility (HMG) box A, box B, and C-terminal acidic regions. In this study, a recombinant TAT linked HMGB1 box A (rTAT-HMGB1A) peptide was expressed, purified, and characterized as a carrier of nucleic acids. The HMGB1A cDNA was amplified by PCR, and cloned into the pET21a expression vector with the TAT domain located at the N-terminus. The rTAT-HMGB1A peptide was overexpressed and purified using Nickel affinity chromatography. A recombinant HMGB1A (rHMGB1A) peptide without the TAT domain was also overexpressed and purified as a control. In gel retardation assays, both the rHMGB1A and rTAT-HMGB1A peptides formed complexes with DNA equally well. However, transfection assays showed that the rTAT-HMGB1A peptide had a higher gene transfer efficiency than rHMGB1A. Finally, rTAT-HMGB1A had no cytotoxicity to HEK 293 cells suggesting that rTAT-HMGB1A may be useful as a non-toxic gene delivery carrier.     

Thermodynamics of HMGB1 interaction with duplex DNA

The high mobility group protein HMGB1 is a small, highly abundant protein that binds to DNA in a non-sequence-specific manner. HMGB1 consists of 2 DNA binding domains, the HMG boxes A and B, followed by a short basic region and a continuous stretch of 30 glutamate or aspartate residues. Isothermal titration calorimetry was used to characterize the binding of HMGB1 to the double-stranded model DNAs poly(dAdT).(dTdA) and poly(dGdC).(dCdG). To elucidate the contribution of the different structural motifs to DNA binding, calorimetric measurements were performed comparing the single boxes A and B, the two boxes plus or minus the basic sequence stretch (AB(bt) and AB), and the full-length HMGB1 protein. Thermodynamically, binding of HMGB1 and all truncated constructs to duplex DNA was characterized by a positive enthalpy change at 15 degrees C. From the slopes of the temperature dependence of the binding enthalpies, heat capacity changes of -0.129 +/- 0.02 and -0.105 +/- 0.05 kcal mol(-1) K(-1) were determined for box A and full-length HMGB1, respectively. Significant differences in the binding characteristics were observed using full-length HMGB1, suggesting an important role for the acid tail in modulating DNA binding. Moreover, full-length HMGB1 binds differently these two DNA templates: binding to poly(dAdT).(dTdA) was cooperative, had a larger apparent binding site size, and proceeded with a much larger unfavorable binding enthalpy than binding to poly(dGdC).(dCdG).     

HMGB1 protein inhibits DNA replication in vitro: a role of the acetylation and the acidic tail

The high mobility group box (HMGB) 1 protein is a very abundant and conserved protein that is implicated in many key cellular events but its functions within the nucleus remain elusive. The role of this protein in replication of closed circular DNA containing a eukaryotic origin of replication has been studied in vitro by using native and recombinant HMGB1 as well as various modified HMGB1 preparations such as truncated protein, lacking its C-terminal tail, in vivo acetylated protein, and recombinant HMGB1 phosphorylated in vitro by protein kinase C (PKC). Native HMGB1 extracted from tumour cells inhibits replication and this effect is reduced upon acetylation and completely abolished upon removal of the acidic C-terminal tail. Recombinant HMGB1, however, fails to inhibit replication but it acquires such a property following in vitro phosphorylation by PKC.