Pigaleva, M. | bioRxiv Science

bioRxiv2026

Thermodynamic properties and stability of HMGB1 complexes with linear polyelectrolytes elucidated by nano differential scanning fluorimetry

The High Mobility Group Box 1 (HMGB1) protein performs multiple essential functions in the body, ranging from DNA regulation to the activation and mediation of immune responses. However, HMGB1 has been also implicated in several pathological conditions, such as rheumatoid arthritis, sepsis, autoimmune diseases, tumors, and Alzheimer's disease. As a result, HMGB1 is of increasing interest as a therapeutic target. Binding to heparin has been reported to inhibit HMGB1's pathological activity during sepsis in clinical settings. In this work, we compare the interactions of HMGB1 with heparin and its' synthetic analog linear polyglycerol sulfate (lPGS) from the viewpoint of stability and changes to association behavior. This analysis focuses on thermal stability, secondary-structure changes, and particle-size evolution using nano-differential scanning fluorimetry (nanoDSF), circular dichroism spectroscopy (CD), and dynamic light scattering (DLS).

biochemistry↗

bioRxiv2025

HMGB1 B-box domain self-complexes promote protein-polyelectrolyte interactions.

HMGB1, a nuclear DNA-binding protein, can be secreted by activated immune cells or passively released from damaged cells. In such cases, HMGB1 functions as an alarmin that activates the immune system. Excessive inflammation may lead to pathogenesis, whereas this response can be dampened by polyanion binding, which impedes further receptor recognition. Moreover, HMGB1 is known to form liquid droplets in the cellular environment--a phase separation directly linked to its proper function. While the A-Box domain is believed to be primarily responsible for heparin binding due to its conserved binding site, the association and phase separation behavior of HMGB1 may be mediated by the B-box domain, owing to its extended hydrophobic regions. In this study, we first demonstrated that the B-box protein forms 30-nm large self-associates while maintaining its structure. Next, using molecularly sensitive EPR spectroscopy, we showed that the presence of these protein associations significantly enhances interactions with heparin. Notably, the local conformational changes induced by heparin are similar in both individual protein chains and their self-associated forms. To explain this effect, AlphaFold modeling was employed, revealing that the formation of protein multimers induces charge redistribution, resulting in an extended positively charged region that enhances electrostatic attraction to negatively charged polyanions, such as heparin.

biochemistry↗