The DNA sequence specificity of HMG boxes lies in the minor wing of the structure.

To establish the basis of sequence-specific DNA recognition by HMG boxes we separately transferred the minor and major wings from the sequence-specific HMG box of TCF1 alpha into their equivalent position in the non-sequence-specific box 2 of HMG1. Thus chimera THT1 contains the minor wing (of 11 N-terminal and 25 C-terminal residues) from the HMG box of TCF1 alpha and the major wing (the 45 residue central section) from HMG1 box 2, whilst the situation is reversed in chimera HTH1. The structural integrity of the two chimeric proteins was established by CD, NMR and their binding to four-way junction DNA. Gel retardation and circular permutation assays showed that only chimera THT1, containing the TCF1 alpha minor wing, formed a sequence-specific complex and bent the DNA. The bend angle was estimated to be 59 degrees for chimera THT1 and 52 degrees for the HMG box of TCF1 alpha. Our results, in combination with mutagenesis and other data, suggests a model for the DNA binding of HMG boxes in which the N-terminal residues and part of helix 1 contact the minor groove on the outside of a bent DNA duplex.     

DNA binding by single HMG box model proteins

The HMG1/2 family is a large group of proteins that share a conserved sequence of ~80 amino acids rich in basic, aromatic and proline side chains, referred to as an HMG box. Previous studies show that HMG boxes can bind to DNA in a structure-specific manner. To define the basis for DNA recognition by HMG boxes, we characterize the interaction of two model HMG boxes, one a structure-specific box, rHMGb from the rat HMG1 protein, the other a sequence-specific box, Rox1 from yeast, with oligodeoxynucleotide substrates. Both proteins interact with single-stranded oligonucleotides in this study to form 1:1 complexes. The stoichiometry of binding of rHMGb to duplex or branched DNAs differs: for a 16mer duplex we find a weak 2:1 complex, while a 4:1 protein:DNA complex is detected with a four-way DNA junction of 16mers in the presence of Mg²⁺. In the case of the sequence-specific Rox1 protein we find tight 1:1 and 2:1 complexes with its cognate duplex sequence and again a 4:1 complex with four-way branched DNA. If the DNA branching is reduced to three arms, both proteins form 3:1 complexes. We believe that these multimeric complexes are relevant for HMG1/2 proteins in vivo, since Mg²⁺ is present in the nucleus and these proteins are expressed at a very high level.     

Investigation of the structural stability of hUBF HMG box 5 by native-state hydrogen exchange

HMG box 5 of human upstream binding factor (hUBF) consists of three alpha-helices arranged in an L-shape with a hydrophobic core embraced by these helices and stabilized by extensive hydrophobic interactions between nonpolar residues around the core. The GdmCl-induced equilibrium unfolding transition of HMG box 5 of hUBF was monitored by both circular dichroism (CD) and fluorescence spectra. A cooperative two-state unfolding process was observed. The unfolding free energy, DeltaGU(D2O), and the cooperativity of the unfolding reaction, m, are 4.6 +/- 0.16 kcal x mol-1 and 1.62 +/- 0.06 kcal x mol-1 x M-1, respectively. Native-state hydrogen exchange (NHX) experiments under EX2 conditions were performed. NHX results clearly show that the hydrophobic core among the three helices is a slow-exchange core. The three helices would not contribute equally to the stability of the native protein. Helix 3 appears to contribute the least to the stability. The NHX data have also allowed the local, subglobal, and global unfolding structures of hUBF HMG box 5 to be dissected, and common global and subglobal unfolding units were successfully detected.     

Solution structure and backbone dynamics of the DNA-binding domain of mouse Sox-5

The fold of the murine Sox-5 (mSox-5) HMG box in free solution has been determined by multidimensional NMR using (15)N-labeled protein and has been found to adopt the characteristic twisted L-shape made up of two wings: the major wing comprising helix 1 (F10--F25) and helix 2 (N32--A43), the minor wing comprising helix 3 (P51--Y67) in weak antiparallel association with the N-terminal extended segment. (15)N relaxation measurements show considerable mobility (reduced order parameter, S(2)) in the minor wing that increases toward the amino and carboxy termini of the chain. The mobility of residues C-terminal to Q62 is significantly greater than the equivalent residues of non-sequence-specific boxes, and these residues show a weaker association with the extended N-terminal segment than in non-sequence boxes. Comparison with previously determined structures of HMG boxes both in free solution and complexed with DNA shows close similarity in the packing of the hydrophobic cores and the relative disposition of the three helices. Only in hSRY/DNA does the arrangement of aromatic sidechains differ significantly from that of mSox-5, and only in rHMG1 box 1 bound to cisplatinated DNA does helix 1 have no kink. Helix 3 in mSox-5 is terminated by P68, a conserved residue in DNA sequence-specific HMG boxes, which results in the chain turning through approximately 90 degrees.     

Structure-specific binding of the two tandem HMG boxes of HMG1 to four-way junction DNA is mediated by the A domain

We have investigated the nature of the "structure-specific" binding of the tandem A and B HMG boxes of high mobility group protein 1 (HMG1) to four-way junction DNA. AB didomain binding favours the open, planar form of the junction, as shown by reaction with potassium permanganate. Site-directed cleavage of the DNA by a 1, 10-phenanthroline-copper moiety attached to unique natural or engineered cysteine residues in the A or B domain shows that the two linked HMG boxes are not functionally equivalent in four-way junction binding. The A domain of the didomain binds to the centre of the junction, mediating structure-specific binding; the concave surface of the domain interacts with the widened minor groove at the centre, contacting one of the four strands of the junction, and the short arm comprising helices I and II and the connecting loop protrudes into the central hole. The B domain makes contacts along one of the arms, presumably stabilising the binding of the didomain through additional non-sequence-specific interactions. The isolated B domain can, however, bind to the centre of the junction. The preferential binding of the A domain of the AB didomain to the centre correlates with our previous finding of a higher preference of the isolated A domain than of the B domain for this structurally distinct DNA ligand. It is probably at least partly due to the higher positive surface potential in the DNA-binding region of the A domain (in particular to an array of positively charged side-chains suitably positioned to interact with the negatively charged phosphates surrounding the central hole of the junction) and partly to differences in residues corresponding to those that intercalate between bases in other HMG box/DNA complexes.     

Temperature‐induced partially unfolded state of hUBF HMG Box‐5: Conformational and dynamic investigations of the Box‐5 thermal intermediate ensemble

Human upstream binding factor (hUBF) HMG Box‐5 is a highly conserved protein domain, containing 84 amino acids and belonging to the family of the nonspecific DNA‐binding HMG boxes. Its native structure adopts a twisted L shape, which consists of three α‐helices and two hydrophobic cores: the major wing and the minor wing. In this article, we report a reversible three‐state thermal unfolding equilibrium of hUBF HMG Box‐5, which is investigated by differential scanning calorimetry (DSC), circular dichroism spectroscopy, fluorescence spectroscopy, and NMR spectroscopy. DSC data show that Box‐5 unfolds reversibly in two separate stages. Spectroscopic analyses suggest that different structural elements exhibit noncooperative transitions during the unfolding process and that the major form of the Box‐5 thermal intermediate ensemble at 55°C shows partially unfolded characteristics. Compared with previous thermal stability studies of other boxes, it appears that Box‐5 possesses a more stable major wing and two well separated subdomains. NMR chemical shift index and sequential ¹H^N_i‐¹H^N_i+1 NOE analyses indicate that helices 1 and 2 are native‐like in the thermal intermediate ensemble, while helix 3 is partially unfolded. Detailed NMR relaxation dynamics are compared between the native state and the intermediate ensemble. Our results implicate a fluid helix‐turn‐helix folding model of Box‐5, where helices 1 and 2 potentially form the helix 1‐turn‐helix 2 motif in the intermediate, while helix 3 is consolidated only as two hydrophobic cores form to stabilize the native structure. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

A role of basic residues and the putative intercalating phenylalanine of the HMG-1 box B in DNA supercoiling and binding to four-way DNA junctions

HMG (high mobility group) 1 is a chromosomal protein with two homologous DNA-binding domains, the HMG boxes A and B. HMG-1, like its individual HMG boxes, can recognize structural distortion of DNA, such as four-way DNA junctions (4WJs), that are very likely to have features common to their natural, yet unknown, cellular binding targets. HMG-1 can also bend/loop DNA and introduce negative supercoils in the presence of topoisomerase I in topologically closed DNAs. Results of our gel shift assays demonstrate that mutation of Arg(97) within the extended N-terminal strand of the B domain significantly (>50-fold) decreases affinity of the HMG box for 4WJs and alters the mode of binding without changing the structural specificity for 4WJs. Several basic amino acids of the extended N-terminal strand (Lys(96)/Arg(97)) and helix I (Arg(110)/Lys(114)) of the B domain participate in DNA binding and supercoiling. The putative intercalating hydrophobic Phe(103) of helix I is important for DNA supercoiling but dispensable for binding to supercoiled DNA and 4WJs. We conclude that the B domain of HMG-1 can tolerate substitutions of a number of amino acid residues without abolishing the structure-specific recognition of 4WJs, whereas mutations of most of these residues severely impair the topoisomerase I-mediated DNA supercoiling and change the sign of supercoiling from negative to positive.     

Solution structure of a DNA-binding domain from HMG1.

We have determined the tertiary structure of box 2 from hamster HMG1 using bacterial expression and 3D NMR. The all alpha-helical fold is in the form of a V-shaped arrowhead with helices along two edges and one rather flat face. This architecture is not related to any of the known DNA binding motifs. Inspection of the fold shows that the majority of conserved residue positions in the HMG box family are those involved in maintaining the tertiary structure and thus all homologous HMG boxes probably have essentially the same fold. Knowledge of the tertiary structure permits an interpretation of the mutations in HMG boxes known to abrogate DNA binding and suggests a mode of interaction with bent and 4-way junction DNA.     

The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins.

The mammalian nuclear protein HMG1 contains two segments that show a high sequence similarity to each other. Each of the segments, produced separately from the rest of the protein in Escherichia coli, binds to DNA with high specificity: four-way junction DNA of various sequences is bound efficiently, but linear duplex DNA is not. Both isolated segments exists as dimers in solution, as shown by gel filtration and chemical crosslinking experiments. HMG1-like proteins are present in yeast and in protozoa: they consist of a single repetition of a motif extremely similar to the DNA binding segments of HMG1, suggesting that they too might form dimers with structural specificity in DNA binding. Sequences with recognizable similarity to either of the two DNA binding segments of HMG1, called HMG boxes, also occur in a few eukaryotic regulatory proteins. However, these proteins are reported to bind to specific sequences, suggesting that the HMG box of proteins distantly related to HMG1 might differ significantly from the HMG box of HMG1-like proteins.     

Structure of the HMG box motif in the B-domain of HMG1.

The conserved, abundant chromosomal protein HMG1 consists of two highly homologous, folded, basic DNA-binding domains, each of approximately 80 amino acid residues, and an acidic C-terminal tail. Each folded domain represents an 'HMG box', a sequence motif recently recognized in certain sequence-specific DNA-binding proteins and which also occurs in abundant HMG1-like proteins that bind to DNA without sequence specificity. The HMG box is defined by a set of highly conserved residues (most distinctively aromatic and basic) and appears to define a novel DNA-binding structural motif. We have expressed the HMG box region of the B-domain of rat HMG1 (residues 88-164 of the intact protein) in Escherichia coli and we describe here the determination of its structure by 2D 1H-NMR spectroscopy. There are three alpha-helices (residues 13-29, 34-48 and 50-74), which together account for approximately 75% of the total residues and contain many of the conserved basic and aromatic residues. Strikingly, the molecule is L-shaped, the angle of approximately 80 degrees between the two arms being defined by a cluster of conserved, predominantly aromatic, residues. The distinctive shape of the HMG box motif, which is distinct from hitherto characterized DNA-binding motifs, may be significant in relation to its recognition of four-way DNA junctions.     

Structural requirements for cooperative binding of HMG1 to DNA minicircles

DNA minicircles, where the length of DNA is below the persistence length, are highly effective, preferred, ligands for HMG-box proteins. The proteins bind to them "structure-specifically" with affinities in the nanomolar range, presumably to an exposed widened minor groove. To understand better the basis of this preference, we have studied the binding of HMG1 (which has two tandem HMG boxes linked by a basic extension to a long acidic tail) and Drosophila HMG-D (one HMG box linked by a basic region to a short and less acidic tail), and their HMG-box domains, to 88 bp and 75 bp DNA minicircles. In some cases we see cooperative binding of two molecules to the circles. The requirements for strong cooperativity are two HMG boxes and the basic extension; the latter also appears to stabilize and constrain the complex, preventing binding of further protein molecules. HMG-D, with a single HMG box, does not bind cooperatively. In the case of HMG1, the acidic tail is not required for cooperativity and does not affect binding significantly, in contrast to a much greater effect with linear DNA, or even four-way junctions (another distorted DNA substrate). Such effects could be relevant in the hierarchy of binding of HMG-box proteins to DNA distortions in vivo, where both single-box and two-box proteins might co-exist, with or without basic extensions and acidic tails.     

Interactions between HMG boxes.

Many proteins consist of subdomains that can fold and function independently. We investigate here the interaction between the two high mobility group (HMG) box subdomains of the nuclear protein rHMG1. An HMG box is a conserved amino acid sequence of approximately 80 amino acids rich in basic, aromatic and proline side chains that is active in binding DNA in a sequence or structure-specific manner. In the case of HMG1, each box can bind structural DNA substrates including four-way junctions (4WJs) and branched or kinked DNA duplexes. Since proteins containing up to six HMG boxes are known, the question arises whether linking subdomains together influences the folding or function of individual boxes. In an effort to understand interactions between individual DNA-binding domains in HMG1, we created new fusion proteins: one is an inversion of the order of the AB di-domain in HMG1 (BA); in the second, we added a third A domain C-terminal to the AB di-domain (ABA). Pairs of boxes, AB or BA, behave similarly and are functionally active. By contrast, the ABA triple subdomain construct is partially unfolded and is less active than individual boxes or di-domains. Thus, long-range inter-domain effects can influence the activity of HMG boxes.     

Recognition of distorted DNA structures by HMG domains

Recent biochemical and structural studies have shown that the preferential recognition of distorted DNA structures, including DNA bulges, four-way junctions and cis-platinated DNA, by HMG domains is dependent on residues immediately preceding the second alpha helix of the L-shaped HMG domain.