Role of the Acidic Tail of High Mobility Group Protein B1 (HMGB1) in Protein Stability and DNA Bending

Abstract

High mobility group box (HMGB) proteins are abundant nonhistone proteins found in all eukaryotic nuclei and are capable of binding/bending DNA. The human HMGB1 is composed of two binding motifs, known as Boxes A and B, are L-shaped alpha-helix structures, followed by a random-coil acidic tail that consists of 30 Asp and Glu residues. This work aimed at evaluating the role of the acidic tail of human HMGB1 in protein stability and DNA interactions. For this purpose, we cloned, expressed and purified HMGB1 and its tailless form, HMGB1ΔC, in E. coli strain. Tryptophan fluorescence spectroscopy and circular dichroism (CD) experiments clearly showed an increase in protein stability promoted by the acidic tail under different conditions, such as the presence of the chemical denaturant guanidine hydrochloride (Gdn.HCl), high temperature and low pH. Folding intermediates found at low pH for both proteins were denatured only in the presence of chemical denaturant, thus showing a relatively high stability. The acidic tail did not alter the DNA-binding properties of the protein, although it enhanced the DNA bending capability from 76° (HMGB1ΔC) to 91° (HMGB1), as measured using the fluorescence resonance energy transfer technique. A model of DNA bending in vivo was proposed, which might help to explain the interaction of HMGB1 with DNA and other proteins, i.e., histones, and the role of that protein in chromatin remodeling.

The HMGB1 C-Terminal Tail Regulates DNA Bending

“…In circularization assays the AB protein bent DNA more effectively than either of the single HMG box domains [18,19], and the full length protein at low concentrations [18]. In contrast to our findings, previous ensemble FRET experiments found that the AB protein bent DNA to a lesser extent than full length HMGB1 [24]. It is possible, however, that the DNA in these experiments was not fully occupied by the AB protein; therefore the measured FRET would be the average of the FRET from unbound DNA and bent DNA in the population, resulting in an apparent lower level of bending for the population.…”

“…Dual-laser optical tweezer experiments showed that the linked A and B boxes of HMGB1 bend DNA to a somewhat lesser degree than a single HMG box [22], and similar results were obtained using atomic force microscopy [23]. Other studies have shown that deleting the acidic C-terminal tail from full length HMGB1 can enhance bending [18], while others reported that deleting the tail reduces bending [24]. Thus, the mechanism by which HMGB1 bends DNA, and the relative contributions of the A box, B box, and C-terminal tail to bending are not completely clear.…”

The Role of High Mobility Group Box 1 in Innate Immunity

“…Another peptide in the B box (130-139) is responsible for promoting erythroleukemia cell differentiation, where the presence of extracellular HMGB1 is essential in erythroid differentiation 21. The C-terminal tail is responsible for DNA damage repair and DNA bending, which then promotes other transcription factors to bind DNA 22,23. The significance of this C-terminal tail lies on its role in increasing the structural stability of HMGB1 and binding to A and B box alternatively 24,25.…”

Nucleic acid scavenging microfiber mesh inhibits trauma-induced inflammation and thrombosis

“…NABPs immobilized on PSMA/polystyrene microfiber meshes inhibited negatively charged polymeric molecules such as DNA, RNA and heparan sulfate [41], but did not inhibit Pam3CSK4, a cationic amphiphilic lipopeptide, and anionic metabolic products (e.g., ATP and uric acid). Furthermore, the NABP-immobilized microfiber meshes captured and removed circulating HMGB1, a DNA-binding protein composed of two positively charged DNA-binding motifs and a C-terminal acidic tail [42]. This highly negatively charged C-terminal domain likely binds to NABPs.…”