Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding

NHP6A is a chromatin-associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non-specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR-based model structure of the DNA complex. The free NHP6A folds into an L-shaped three alpha-helix structure, and contains an unstructured 17 amino acid basic tail N-terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C,15N-labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY-DNA complex. In the model structure of the NHP6A-DNA complex, the N-terminal basic tail is wrapped around the major groove in a manner mimicking the C-terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A-DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.     

Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals their ability to form highly stable DNA-protein complexes

The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb lambda-DNA molecules is investigated using DNA micromanipulation. As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending. Most significantly, we find that for each of HMGB1, NHP6A, and HU there is a well-defined protein concentration, not far above the binding threshold, above which the proteins do not spontaneously dissociate. In this regime, the amount of protein bound to the DNA, as assayed by the degree to which the DNA is compacted, is unperturbed either by replacing the surrounding protein solution with protein-free buffer or by straightening of the molecule by applied force. Thus, the stability of the protein-DNA complexes formed is dependent on the protein concentration during the binding. HU is distinguished by a switch to a DNA-stiffening function at the protein concentration where the formation of highly stable complexes occurs. Finally, introduction of competitor DNA fragments into the surrounding solution disassembles the stable DNA complexes with HMGB1, NHP6A, and HU within seconds. Since spontaneous dissociation of protein does not occur on a time scale of hours, we conclude that this rapid protein exchange in the presence of competitor DNA must occur only via "direct" DNA-DNA contact. We therefore observe that protein transport along DNA by direct transfers occurs even for proteins such as NHP6A and HU that have only one DNA-binding domain.     

Determining the DNA bending angle induced by non-specific high mobility group-1 (HMG-1) proteins: a novel method

To study the DNA bending induced by non-sequence-specific HMG-1 domain proteins, we have engineered a fusion protein linking the yeast NHP6A with a sequence-specific DNA binding domain, the DNA binding domain of the Hin recombinase, Hin-DBD. A series of biochemical experiments were carried out to characterize the DNA binding property of this fusion protein. Our data showed that the fusion protein not only specifically recognizes a DNA fragment containing the Hin-DBD binding site, but also binds DNA with a higher affinity in comparison with either domain alone. Both domains of the fusion protein are bound to the DNA in juxtaposition. Permutation assays showed that the fusion protein induced a DNA bending at the site of NHP6A binding by an estimated value of 63 degrees. We believe that this experimental design provides an effective vehicle to determine the DNA bending induced by nonspecific HMG-1 proteins.     

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.     

Recognition of DNA interstrand cross-link of antitumor cisplatin by HMGB1 protein

Several proteins that specifically bind to DNA modified by cisplatin, including those containing HMG-domains, mediate antitumor activity of this drug. Oligodeoxyribonucleotide duplexes containing a single, site-specific interstrand cross-link of cisplatin were probed for recognition by the rat chromosomal protein HMGB1 and its domains A and B using the electrophoretic mobility-shift assay. It has been found that the full-length HMGB1 protein and its domain B to which the lysine-rich region (seven amino acid residues) of the A/B linker is attached at the N-terminus (the domain HMGB1b7) specifically recognize DNA interstrand cross-linked by cisplatin. The affinity of these proteins to the interstrand cross-link of cisplatin is not very different from that to the major 1,2-GG intrastrand cross-link of this drug. In contrast, no recognition of the interstrand cross-link by the domain B lacking this region or by the domain A with or without this lysine-rich region attached to its C-terminus is noticed under conditions when these proteins readily bind to 1,2-GG intrastrand adduct. A structural model for the complex formed between the interstrand cross-linked DNA and the domain HMGB1b7 was constructed and refined using molecular mechanics and molecular dynamics techniques. The calculated accessible areas around the deoxyribose protons correlate well with the experimental hydroxyl radical footprint. The model suggests that the only major adaptation necessary for obtaining excellent surface complementarity is extra DNA unwinding (approximately 40 degrees ) at the site of the cross-link. The model structure is consistent with the hypothesis that the enhancement of binding affinity afforded by the basic lysine-rich A/B linker is a consequence of its tight binding to the sugar-phosphate backbone of both DNA strands.     

The DNA architectural protein HMGB1 displays two distinct modes of action that promote enhanceosome assembly

HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.     

The Saccharomyces cerevisiae high mobility group box protein HMO1 contains two functional DNA binding domains

High mobility group box (HMGB) proteins are architectural proteins whose HMG DNA binding domains confer significant preference for distorted DNA, such as 4-way junctions. HMO1 is one of 10 Saccharomyces cerevisiae HMGB proteins, and it is required for normal growth and plasmid maintenance and for regulating the susceptibility of yeast chromatin to nuclease. Using electrophoretic mobility shift assays, we have shown here that HMO1 binds 26-bp duplex DNA with K(d) = 39.6 +/- 5.0 nm and that its divergent box A domain participates in DNA interactions, albeit with low affinity. HMO1 has only modest preference for DNA with altered conformations, including DNA with nicks, gaps, overhangs, or loops, as well as for 4-way junction structures and supercoiled DNA. HMO1 binds 4-way junctions with half-maximal saturation of 19.6 +/- 2.2 nm, with only a modest increase in affinity in the absence of magnesium ions (half-maximal saturation 6.1 +/- 1.1 nm). Whereas the box A domain contributes modest structure-specific binding, the box B domain is required for high affinity binding. HMO1 bends DNA, as measured by DNA cyclization assays, facilitating cyclization of 136-, 105-, and 87-bp DNA, but not 75-bp DNA, and it has a significantly longer residence time on DNA minicircles compared with linear duplex DNA. The unique DNA binding properties of HMO1 are consistent with global roles in the maintenance of chromatin structure.     

Structure of the major single-stranded DNA-binding domain of replication protein A suggests a dynamic mechanism for DNA binding

Although structures of single-stranded (ss)DNA-binding proteins (SSBs) have been reported with and without ssDNA, the mechanism of ssDNA binding in eukarya remains speculative. Here we report a 2.5 Angstroms structure of the ssDNA-binding domain of human replication protein A (RPA) (eukaryotic SSB), for which we previously reported a structure in complex with ssDNA. A comparison of free and bound forms of RPA revealed that ssDNA binding is associated with a major reorientation between, and significant conformational changes within, the structural modules--OB-folds--which comprise the DNA-binding domain. Two OB-folds, whose tandem orientation was stabilized by the presence of DNA, adopted multiple orientations in its absence. Within the OB-folds, extended loops implicated in DNA binding significantly changed conformation in the absence of DNA. Analysis of intermolecular contacts suggested the possibility that other RPA molecules and/or other proteins could compete with DNA for the same binding site. Using this mechanism, protein-protein interactions can regulate, and/or be regulated by DNA binding. Combined with available biochemical data, this structure also suggested a dynamic model for the DNA-binding mechanism.     

Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently

We analyzed the DNA-binding and DNA-bending properties of recombinant HMGB1 proteins based on a rice HMGB1 cDNA. Electrophoretic mobility shift assay demonstrated that rice HMGB1 can bind synthetic four-way junction (4H) DNA and DNA minicircles efficiently but the binding to 4H can be completed out by HMGA and histone H1. Conformational changes were detected by circular dichroism analysis with 4H DNA bound to various concentrations of HMGB1 or its truncated forms. T4 ligase-mediated circularization assays with short DNA fragments of 123 bp showed that the protein is capable of increasing DNA flexibility. The 123-bp DNA formed closed circular monomers efficiently in its presence, similar to that in an earlier study on maize HMG. Additionally, our results show for the first time that the basic N-terminal domain enhances the affinity of the plant HMGB1 protein for 4H DNA, while the acidic C-terminal domain has the converse effects.     

Redox state-dependent interaction of HMGB1 and cisplatin-modified DNA

HMGB1, one of the most abundant nuclear proteins, has a strong binding affinity for cisplatin-modified DNA. It has been proposed that HMGB1 enhances the anticancer efficacy of cisplatin by shielding platinated DNA lesions from repair. Two cysteine residues in HMGB1 domain A form a reversible disulfide bond under mildly oxidizing conditions. The reduced domain A protein binds to a 25-bp DNA probe containing a central 1,2-d(GpG) intrastrand cross-link, the major platinum-DNA adduct, with a 10-fold greater binding affinity than the oxidized domain A. The binding affinities of singly and doubly mutated HMGB1 domain A, respectively deficient in one or both cysteine residues that form the disulfide bond, are unaffected by changes in external redox conditions. The redox-dependent nature of the binding of HMGB1 domain A to cisplatin-modified DNA suggests that formation of the intradomain disulfide bond induces a conformational change that disfavors binding to cisplatin-modified DNA. Hydroxyl radical footprinting analyses of wild-type domain A bound to platinated DNA under different redox conditions revealed identical cleavage patterns, implying that the asymmetric binding mode of the protein across from the platinated lesion is conserved irrespective of the redox state. The results of this study reveal that the cellular redox environment can influence the interaction of HMGB1 with the platinated DNA and suggest that the redox state of the A domain is a potential factor in regulating the role of the protein in modulating the activity of cisplatin as an anticancer drug.     

Mechanism of high-mobility group protein B enhancement of progesterone receptor sequence-specific DNA binding

The DNA-binding domain (DBD) of progesterone receptor (PR) is bipartite containing a zinc module core that interacts with progesterone response elements (PRE), and a short flexible carboxyl terminal extension (CTE) that interacts with the minor groove flanking the PRE. The chromosomal high-mobility group B proteins (HMGB), defined as DNA architectural proteins capable of bending DNA, also function as auxiliary factors that increase the DNA-binding affinity of PR and other steroid receptors by mechanisms that are not well defined. Here we show that the CTE of PR contains a specific binding site for HMGB that is required for stimulation of PR-PRE binding, whereas the DNA architectural properties of HMGB are dispensable. Specific PRE DNA inhibited HMGB binding to the CTE, indicating that DNA and HMGB–CTE interactions are mutually exclusive. Exogenous CTE peptide increased PR-binding affinity for PRE as did deletion of the CTE. In a PR-binding site selection assay, A/T sequences flanking the PRE were enriched by HMGB, indicating that PR DNA-binding specificity is also altered by HMGB. We conclude that a transient HMGB–CTE interaction alters a repressive conformation of the flexible CTE enabling it to bind to preferred sequences flanking the PRE.     

Structure of DNA complexes with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions: 1. Circular dichroism spectroscopy

Mechanisms of interaction of DNA with nonhistone chromosomal protein HMGB1 and linker histone H1 have been studied by means of circular dichroism and absorption spectroscopy. Both proteins are located in the internucleosomal regions of chromatin. It is demonstrated that the properties of DNA-protein complexes depend on the protein content and cannot be considered as a mere summing up of the effects of individual protein components. Interaction of the HMGB1 and H1 proteins is shown with DNA to be cooperative rather than competitive. Lysine-rich histone H1 facilitates the binding of HMGB1 to DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino acid residues in the C-terminal domain of HMGB1. The observed joint action of HMGB1 and H1 stimulates DNA condensation with the formation of anisotropic DNA-protein complexes with typical ψ-type CD spectra. Structural organization of the complexes depends not only on DNA-protein interactions but also on interaction between the HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the mode of interactions between components in the triple DNA-HMGB1-H1 complex. The binding of Mn²⁺ ions weakens DNA-protein interactions and strengthens protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.     

Determinants of HMGB proteins required to promote RAG1/2-recombination signal sequence complex assembly and catalysis during V(D)J recombination

Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.     

Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ?29 DNA mimic protein p56

Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage ϕ29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.     

The structure of the complexes of DNA with non-histone chromosomal protein HMGB1 in the presence of manganese ions

The complexes of DNA - HMGB1 protein - manganese ions have been studied using circular dichroism (CD) technique. It was shown that in such three-component system the interactions of both the protein and metal ions with DNA differ from those in two-component complexes. The manganese ions do not affect the CD spectrum of free HMGB1 protein. However, Mn2+ ions induce considerable changes in the CD spectrum of free DNA in the spectral range of 260-290 nm. The presence of Mn2+ ions prevents formation of the ordered supramolecular structures specific for the HMGB1-DNA complexes. The interaction of manganese ions with DNA has a marked influence on the local DNA structure changing the properties of protein-binding sites. This results in the serious decrease in cooperativity of the DNA-protein binding. Such changes in the mode of the DNA-protein interactions occur at concentrations as small as 0.01 mM Mn2+. Moreover, the changes in local DNA structure induced by manganese ions promote the appearance of new HMGB1 binding sites on the DNA double helix. At the same time interactions with HMGB1 protein induce alterations in the structure of the DNA double helix which increase with a growth of the protein/DNA ratio. These alterations make the DNA/protein complex especially sensitive to manganese ions. Under these conditions the Mn2+ ions strongly affect the DNA structure that reflects in abrupt changes of the CD spectra of DNA in the complex in the range of 260-290 nm. Thus, structural changes of the DNA double helix in the three-component DNA-HMGB1-Mn2+ complexes come as a result of the combined and interdependent interactions of DNA with Mn2+ ions and the molecules of HMGB1.