DNA binding and bending by HMG boxes: energetic determinants of specificity

To clarify the physical basis of DNA binding specificity, the thermodynamic properties and DNA binding and bending abilities of the DNA binding domains (DBDs) of sequence-specific (SS) and non-sequence-specific (NSS) HMG box proteins were studied with various DNA recognition sequences using micro-calorimetric and optical methods. Temperature-induced unfolding of the free DBDs showed that their structure does not represent a single cooperative unit but is subdivided into two (in the case of NSS DBDs) or three (in the case of SS DBDs) sub-domains, which differ in stability. Both types of HMG box, most particularly SS, are partially unfolded even at room temperature but association with DNA results in stabilization and cooperation of all the sub-domains. Binding and bending measurements using fluorescence spectroscopy over a range of ionic strengths, combined with calorimetric data, allowed separation of the electrostatic and non-electrostatic components of the Gibbs energies of DNA binding, yielding their enthalpic and entropic terms and an estimate of their contributions to DNA binding and bending. In all cases electrostatic interactions dominate non-electrostatic in the association of a DBD with DNA. The main difference between SS and NSS complexes is that SS are formed with an enthalpy close to zero and a negative heat capacity effect, while NSS are formed with a very positive enthalpy and a positive heat capacity effect. This indicates that formation of SS HMG box-DNA complexes is specified by extensive van der Waals contacts between apolar groups, i.e. a more tightly packed interface forms than in NSS complexes. The other principal difference is that DNA bending by the NSS DBDs is driven almost entirely by the electrostatic component of the binding energy, while DNA bending by SS DBDs is driven mainly by the non-electrostatic component. The basic extensions of both categories of HMG box play a similar role in DNA binding and bending, making solely electrostatic interactions with the DNA.     

The DNA bend angle and binding affinity of an HMG box increased by the presence of short terminal arms.

The HMG box of human LEF-1 (hLEF-1, formerly TCF1alpha) has been expressed in four forms: a parent box of 81 amino acids and constructs having either a 10 amino acid C-terminal extension, a 9 amino acid N-terminal extension, or both. These four species have been compared for DNA binding and bending ability using a 28 bp recognition sequence from the TCR alpha-chain enhancer. In the bending assay, whereas the parent box and that with the N-terminal extension bent the DNA by 57/58 degrees, the box extended at the C-terminus bent the DNA by 77/78 degrees, irrespective of the presence or absence of the N-terminal extension. A 6- fold increase in DNA affinity also resulted from addition of both terminal extensions. These observations redefine the functional boundaries of the HMG box. The structure of a mouse LEF-1/DNA complex recently published [Love et al. (1995) Nature 376, 791-795] implies that the higher DNA affinity and in particular the increased bend angle observed are consequences, at least in part, of the C-terminal extension spanning the major groove on the inside of the DNA bend.     

HMGB binding to DNA: single and double box motifs

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° 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°, 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.     

HMG box A in HMG2 protein functions as a mediator of DNA structural alteration together with box B

Nonhistone protein HMG2, like HMG1, binds with B-DNA in a sequence-nonspecific manner and causes structural alterations in DNA such as bending, kinking and unwinding. Here, we studied the functions of HMG2 domains in the DNA structural alteration and modulation by using various HMG2 peptides, and we demonstrated several new findings. The HMG box itself as a DNA-binding motif may have the basic function of inducing curvature, resulting in the apparent DNA bending in the DNA cyclization assay, but not of abruptly kinking DNA. The DNA-binding activity of HMG box B, which is enhanced by the presence of box A, together with the flanking regions of box B, causes DNA bending accompanying the kinking of the DNA main chain. The DNA unwinding accompanied by DNA kinking diminishes cruciform structures in supercoiled DNA. Analysis using mutant peptides for box A confirmed that box A in HMG2 functions as a mediator of DNA structural alteration together with box B. The present studies on the functional properties of the respective regions of HMG2 may help to elucidate the protein function.     

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.     

Stopped-flow fluorescence studies of HMG-domain protein binding to cisplatin-modified DNA

High-mobility group (HMG) domain proteins bind specifically to the major DNA adducts formed by the anticancer drug cisplatin and can modulate the biological response to this inorganic compound. Stopped-flow fluorescence studies were performed to investigate the kinetics of formation and dissociation of complexes between HMG-domain proteins and a series of 16-mer oligonucleotide probes containing both a 1,2-intrastrand d(GpG) cisplatin cross-link and a fluorescein-modified deoxyuridine residue. Rate constants, activation parameters, and dissociation constants were determined for complexes formed by HMG1 domain A and the platinated DNA probes. The sequence context of the cisplatin adduct modulates the value of the associative rate constant for HMG1 domain A by a factor of 2-4, contributing significantly to differences in binding affinity. The rates of association or dissociation of the protein-DNA complex were similar for a 71 bp platinated DNA analogue. Additional kinetic studies performed with HMG1 domain B, an F37A domain A mutant, and the full-length HMG1 protein highlight differences in the binding properties of the HMG domains. The stopped-flow studies demonstrate the utility of the fluorescein-dU probe in studying protein-DNA complexes. The kinetic data will assist in determining what role these proteins might play in the cisplatin mechanism of action.     

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.     

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.