DNA-binding properties of the tandem HMG boxes of high-mobility-group protein 1 (HMG1).

High-mobility-group protein 1 (HMG1) is a conserved chromosomal protein with two homologous DNA-binding HMG-box domains, A and B, linked by a short basic region to an acidic carboxy-terminal tail. NMR spectroscopy on the free didomain (AB) shows that the two HMG boxes do not interact. The didomain has a higher affinity for all DNA substrates tested than single HMG-box domains and has a significantly higher ability to distort DNA by bending and supercoiling. The interaction of the didomain with DNA is stabilized by the presence of the basic region (approximately 20 residues, 9 of which are Lys) that links the second HMG box to the acidic tail in intact HMG1; this may be, at least in part, why this region also enhances supercoiling of relaxed circular DNA by the didomain and circularization of short DNA fragments (in the presence of ligase). Competition assays suggest significantly different structure-specific preferences of single and tandem HMG boxes for four-way junction and supercoiled plasmid DNA. Binding to supercoiled DNA appears to be promoted by protein oligomerization, which is pronounced for the didomains. Electron microscopy suggests that the oligomers are globular aggregates, associated with DNA looping. One box versus two (or several) is likely to be an important determinant of the properties of (non-sequence specific) HMG-box proteins.

Structure of the A-domain of HMG1 and its interaction with DNA as studied by heteronuclear three- and four-dimensional NMR spectroscopy.

HMG1 has two homologous, folded DNA-binding domains ("HMG boxes"), A and B, linked by a short basic region to an acidic C-terminal domain. Like the whole protein, which may perform an architectural role in chromatin, the individual boxes bind to DNA without sequence specificity, have a preference for distorted or prebent DNA, and are able to bend DNA and constrain negative superhelical turns. They show qualitatively similar properties with quantitative differences. We have previously determined the structure of the HMG box from the central B-domain (77 residues) by two-dimensional NMR spectroscopy, which showed that it contains a novel fold [Weir et al. (1993) EMBO J. 12, 1311-1319]. We have now determined the structure of the A-domain (as a Cys-->Ser mutant at position 22 to avoid oxidation, without effect on its DNA-binding properties or structure) using heteronuclear three- and four-dimensional NMR spectroscopy. The A-domain has a very similar global fold to the B-domain and the Drosophila protein HMG-D [Jones et al. (1994) Structure 2, 609-627]. There are small differences between A and B, in particular in the orientation of helix I, where the B-domain is more similar to HMG-D than it is to the A-domain; these differences may turn out to be related to the subtle differences in functional properties between the two domains [Teo et al. (1995) Eur. J. Biochem. 230, 943-950] and will be the subject of further investigation. NMR studies of the interaction of the A-domain of HMG1 with a short double-stranded oligonucleotide support the notion that the protein binds via the concave face of the L-shaped structure; extensive contacts with the DNA are made by the N-terminal extended strand, the N-terminus of helix I, and the C-terminus of helix II. These contacts are very similar to those seen in the LEF-1 and SRY-DNA complexes [Love et al. (1995) Nature 376, 791-795; Werner et al. (1995) Cell 81, 705-714].

Backbone dynamics of the A-domain of HMG1 as studied by 15N NMR spectroscopy.

The HMG-box sequence motif (approximately 80 residues) occurs in a number of abundant eukaryotic chromosomal proteins such as HMG1, which binds DNA without sequence specificity, but with "structure specificity", as well as in several sequence-specific transcription factors. HMG1 has two such boxes, A and B, which show approximately 30% sequence identity, and an acidic C-terminal tail. The boxes are responsible for the ability of the protein to bend DNA and bind to bent or distorted DNA. The structure of the HMG box has been determined by NMR spectroscopy for the B-domain of HMG1 [Weir et al. (1993) EMBO J. 12, 1311-1319; Read et al. (1993) Nucleic Acids Res. 21, 3427-3436) and for Drosophila HMG-D (Jones et al. (1994) Structure 2, 609-627]. It has an unusual twisted L-shape, suggesting that the protein might tumble anisotropically in solution. In this paper we report studies of the A-domain from HMG1 using 15N NMR spectroscopy which show that the backbone dynamics of the protein can be described by two different rotational correlation times of 9.0 +/- 0.5 and 10.8 +/- 0.5 ns. We show that the relaxation data can be analyzed by assuming that the protein is a rigid, axially symmetric ellipsoid undergoing anisotropic rotational diffusion; the global rotational diffusion constants, D parallel and D perpendicular, were estimated as 2.47 x 10(7) and 1.49 x 10(7) s-1, respectively. By estimating the angle between the amide bond vectors and the major axis of the rotational diffusion tensor from the family of structures determined by NMR spectroscopy [see accompanying paper, Hardman et al. (1995) Biochemistry 34, 16596-16607], we were able to show that the ellipsoid spectral density equation can reproduce the major features of the 15N T1 and T2 profiles of the three helices in the HMG1 A-domain. The backbone dynamics of the A-domain were then compared with those of the B-domain and the HMG box from HMG-D. This comparison strongly supported the differences observed in the orientation of helix I in the three structures, where the B-domain appears to be more similar to HMG-D than it is to the A-domain. These differences may turn out to be related to subtle differences in the DNA-binding properties of the A- and B-domains of HMG1.

Two mutations in the HMG-box with very different structural consequences provide insights into the nature of binding to four-way junction DNA.

Mutation of the highly conserved tryptophan residue in the A-domain HMG-box of HMG1 largely, but not completely, destroys the protein tertiary structure and abolishes its supercoiling ability, but does not abolish structure-specific DNA binding to four-way junctions. Circular dichroism shows that the protein has some residual alpha-helix (< 10%) and does not re-fold in the presence of DNA. Structure-specific DNA binding might therefore be a property of some primary structure element, for example the N-terminal extended strand, which even in the unfolded protein would be held in a restricted conformation by two, largely trans, X-Pro peptide bonds. However, mutation of P5 or P8 of the A-domain to alanine does not abolish the formation of the (first) complex in a gel retardation assay, which probably arises from binding to the junction cross-over, although the P8 mutation does affect the formation of higher complexes which may arise from binding to the junction arms. Since mutation of P8 in the W49R mutant has no effect on structure-specific junction binding, we propose that some residual alpha-helix in the protein might be involved, implicating this element in the interactions of HMG-boxes generally with DNA.