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

Abstract

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 fifty base pairs 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-boxA-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.

Yeast HMO1: Linker Histone Reinvented

“…Most non-sequence-specific chromatin-associated HMGB proteins, e.g., mammalian HMGB1 to -4, possess two HMGB domains and bind preferentially to non-B-form DNA structures such as four-way junctions and DNA modified by the anticancer agent cisplatin (93,94). Exceptions have been described, for example, in S. cerevisiae and Drosophila melanogaster, which encode single HMG box proteins (NHP6A/B and HMGD, respectively) that bind DNA without a sequence preference (95,96).…”

The high mobility group box: the ultimate utility player of a cell

“…Generally, transcription factors are sequence-specific and contain a single HMG box (Figure 1a, Table 1) [18]. However, non-sequence-specific single HMG boxes exist in Drosophila melanogaster (HMGD) [19], Saccharomyces cerevisiae (NHP6A) [20] (Figure 1a), and humans (PMS1) [21]. Tandem HMG box proteins (Figure 1b, Table 1) include HMGB1 through HMGB4, and mitochondrial transcription factor A (mtTFA/TFAM), among others (reviewed in [10, 22]).…”

“…The HMG-box severely bends and underwinds DNA, using electrostatic and hydrophobic interactions to widen the minor groove and induce a bend towards the major groove. Importantly, HMG-box residues that intercalate DNA aid in stabilizing the distorted DNA structure (reviewed in [19]). The HMG boxes of both sequence-specific and non-sequence-specific proteins typically contain a non-polar DNA intercalating residue in the 1° site at the N terminus of alpha helix 1 (in red) (Figure 1, Figure 2).…”

Two high-mobility group box domains act together to underwind and kink DNA

“…The HMGB1 domains are unequal in these properties: box A recognizes both pre-bent and linear DNA more tightly than box B (Mü ller et al, 2001), but box B binds to mini-circles (Webb et al, 2001) and bends linear DNA to a greater extent than box A (Paull et al, 1993;. This dramatic distortion of DNA is dependent on both shape complementarity and DNA intercalation of two apolar residues (Churchill et al, 2010;Murphy & Churchill, 2000;Roemer et al, 2008). The primary intercalation residue is in helix I (1 in Fig.…”