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

Fabricio S Belgrano,Ronaldo Mohana-Borges,Isabel C De Abreu Da Silva,Francisco M Bastos De Oliveira,Marcelo R Fantappié,Sabato D'Auria

doi:10.1371/journal.pone.0079572

Fabricio S Belgrano, Ronaldo Mohana-Borges + Show 4 more

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https://doi.org/10.1371/journal.pone.0079572

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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.

Highlights

High mobility group box (HMGB) proteins belong to a superfamily of nuclear proteins with DNA-binding capabilities [1]
The purity and identity of High Mobility Group Protein B1 (HMGB1) and HMGB1ΔC were confirmed by 15% SDS-PAGE (Figure 1B) and by western blotting using monoclonal antibody anti-human HMGB1 (Figure 1C), respectively
The secondary and tertiary structures of HMGB1 and HMGB1ΔC were monitored by circular dichroism (CD) and Trp fluorescence spectroscopy, respectively, to assess whether the proteins were properly folded during the purification steps and to determine the effect of the acidic tail on HMGB1-folding

Summary

Introduction

High mobility group box (HMGB) proteins belong to a superfamily of nuclear proteins with DNA-binding capabilities [1]. The human HMGB1 protein is composed of 215 amino acids and is functionally divided into three domains: two positively charged DNA-binding motifs (Boxes A and B) and a C-terminal domain composed of a segment of 30 acidic residues (Figure 1A). The two boxes are structurally similar, comprising 3 α-helices that confer an “L-shaped” DNA-binding domain, with an angle of 80° between the arms [2,3,4,5]. The minor groove of the DNA molecule binds to the concave side of the boxes with no sequence specificity. The current model of action suggests that the HMGB1 protein is capable of binding to and bending DNA randomly, remodeling chromatin in a “hit and run” fashion [6]. HMGB1 has been shown to have high affinity for topologically modified DNA, such as 4-way junctions and kinked, bulged and minicircle DNA [7,8,9,10]

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