Structure of the A-Domain of HMG1 and Its Interaction with DNA as Studied by Heteronuclear Three- and Four-Dimensional NMR Spectroscopy

Abstract

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].

HMGB1 Interacts with Many Apparently Unrelated Proteins by Recognizing Short Amino Acid Sequences

“…This may in turn enrich a particular experiment in a certain set of peptides because of slight changes (in temperature for instance). But it could also be attributable to the differences in sequence of the two HMG boxes because their three-dimensional structures are rather similar (6,8). On the other hand, taking into account sequence identity in HMG boxes A and B is very high when compared with the corresponding domains in HMGB2, it is also very likely that many if not all of the peptide motifs will also be recognized by HMGB2, as well.…”

Mechanisms of DNA binding determined in optical tweezers experiments

“…The structure of an HMG box is shown in Figure 3a. 47 The domain is composed of three helices and a tail, and is stabilized by hydrophobic interactions between neighboring residues. The N-terminal tail is highly variable and may or may not interact with DNA depending on the particular HMGB protein studied.…”

“…41,48 The remaining two helices are known to bind against the minor groove of dsDNA, with hydrophobic side chains from amino acids at one or both of two specific positions intercalating between base pairs, untwisting the duplex and producing a strong bend in the backbone toward the major groove, as shown in the HMGB domain of Figure 3b. [47][48][49][50][51] The angle of this bend has been reported to fall somewhere in the 308 to 1308 range, although values between 708 and 908 are typical for a domain with a single HMG box. 49,[52][53][54][55] HMGB proteins are thought to enable the binding of transcription factors, 50,51,[56][57][58][59] either by binding directly to the factors 60,61 or by stabilizing DNA looping.…”

“…As the protein is added, the persistence length decreases while the contour length increases, changes which are attributed to the intercalation mode described above. [47][48][49] Quantifying the Protein-DNA Interaction. The concentration dependence of the persistence length may yield parameters for the DNA-protein interaction.…”

Co-crystallization and preliminary crystallographic analysis of the high mobility group domain of HMG-D bound to DNA

“…HMG-D contains only a single copy of the HMG-box DNA-binding domain (Ner & Travers, 1994;Wagner et al, 1992) at the N-terminus, which is followed by a`tail' region which has`basic motifs' similar to the C-terminal domain of histone H1 and a C-terminal acidic stretch similar to those in HMG1/2. The structures of the HMG-domain of HMG-D and HMG1 have been determined by NMR (Hardman et al, 1995;Jones et al, 1994;Read et al, 1993;Weir et al, 1993), revealing an L-shaped fold comprised of three helices held together by two hydrophobic cores. The structures of complexes of sequence-speci®c HMG-domains, LEF-1 and SRY, bound to DNA have also been determined by NMR and reveal a common protein fold and severe protein-induced DNA bending toward the major groove (Love et al, 1995;Werner, Bianchi et al, 1995;Werner, Huth et al, 1995).…”