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March 31st, 2025
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Institute of Chemistry and Biochemistry, Freie Universitat Berlin, 14195 Berlin, Germany
biochemistry
biorxiv

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

Kagelmacher, M.Open in Google Scholar•Pigaleva, M.Open in Google Scholar•Zarate, R.Open in Google Scholar•Bechtella, L.Open in Google Scholar•Pagel, K.Open in Google Scholar•Koksch, B.Open in Google Scholar•Dernedde, J.Open in Google Scholar•Hermann, A.Open in Google Scholar•Risse, T.Open in Google Scholar

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.

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