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.     

Mutagenesis analysis of the interaction between the dorsal rel homology domain and HMG boxes of DSP1 protein

DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ((253)KKRK(256)) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.     

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.     

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.     

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.     

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.     

Structural Analysis of HMGD-DNA Complexes Reveals Influence of Intercalation on Sequence Selectivity and DNA Bending

The ubiquitous, eukaryotic, high-mobility group box (HMGB) chromosomal proteins promote many chromatin-mediated cellular activities through their non-sequence-specific binding and bending of DNA. Minor-groove DNA binding by the HMG box results in substantial DNA bending toward the major groove owing to electrostatic interactions, shape complementarity, and DNA intercalation that occurs at two sites. Here, the structures of the complexes formed with DNA by a partially DNA intercalation-deficient mutant of Drosophila melanogaster HMGD have been determined by X-ray crystallography at a resolution of 2.85 Å. The six proteins and 50 bp of DNA in the crystal structure revealed a variety of bound conformations. All of the proteins bound in the minor groove, bridging DNA molecules, presumably because these DNA regions are easily deformed. The loss of the primary site of DNA intercalation decreased overall DNA bending and shape complementarity. However, DNA bending at the secondary site of intercalation was retained and most protein-DNA contacts were preserved. The mode of binding resembles the HMGB1 box A-cisplatin-DNA complex, which also lacks a primary intercalating residue. This study provides new insights into the binding mechanisms used by HMG boxes to recognize varied DNA structures and sequences as well as modulate DNA structure and DNA bending.     

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.     

DNA looping in the RNA polymerase I enhancesome is the result of non-cooperative in-phase bending by two UBF molecules

The so-called upstream binding factor (UBF) is required for the initial step in formation of an RNA polymerase I initiation complex. This function of UBF correlates with its ability to induce the ribosomal enhancesome, a structure which resembles in its mass and DNA content the nucleosome of chromatin. DNA looping in the enhancesome is probably the result of six in-phase bends induced by the HMG boxes of a UBF dimer. Here we show that insertion/deletion mutations in the basic peptide linker lying between the N-terminal dimerisation domain and the first HMG box of Xenopus UBF prevent the DNA looping characteristic of the enhancesome. Using these mutants we demonstrate that (i) the enhancesome structure does not depend on tethering of the entering and exiting DNA duplexes, (ii) UBF monomers induce hemi-enhancesomes, bending the DNA by 175 ± 24° and (iii) two hemi-enhancesomes are precisely phased by UBF dimerisation. We use this and previous data to refine the existing enhancesome model and show that HMG boxes 1 and 2 of UBF lie head-to-head along the DNA.     

DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5.

Sox-5 is one of a family of genes which show homology to the HMG box region of the testis determining gene SRY. We have used indirect immunofluorescence to show that Sox-5 protein is localized to the nucleus of post-meiotic round spermatids in the mouse testis. In vitro footprinting and gel retardation assays demonstrate that Sox-5 binds specifically to the sequence AACAAT with moderately high affinity (Kd of approximately 10(-9) M). Moreover, interaction of Sox-5 with its target DNA induces a significant bend in the DNA, characteristic of HMG box proteins. Circular dichroism spectroscopy of the Sox-5 HMG box and its specific complex with DNA shows an alteration in the DNA spectrum, perhaps as a consequence of DNA bending, but none in the protein spectrum on complex formation. The dependence of the change in the CD spectrum with protein to DNA ratio demonstrates the formation of a 1:1 complex. Analysis of the structure of the Sox-5 HMG box by 2D NMR suggests that both the location of helical secondary structure as well as the tertiary structure is similar to that of HMG1 box 2.