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bioRxiv · 10.1101/2025.11.03.686449

Key determinants of VDAC-hexokinase I complex assembly revealed by a minimal vesicle-based interaction assay

Wessing, M.·Watrinet, I.·Timme, M.·Fiedler, B.·Melo, M. N.·Piehler, J.·Holthuis, J. C. M.
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Abstract

Binding of hexokinase HKI to mitochondrial voltage-dependent anion channels (VDACs) regulates the metabolic fate of glucose and promotes cell survival in hyperglycolytic tumors. Computer simulations indicated that complex assembly relies on intimate contacts between the N-terminal -helix of HKI and a charged bilayer-facing glutamate on the outer wall of VDACs. Protonation of this residue blocks complex formation in silico, explaining the release of HKI from mitochondria observed upon cytosolic acidification. To validate these findings, we here developed an in vitro assay for interrogating HKI binding to VDAC1 reconstituted in vesicles captured on a functionalized surface. TIRF-based quantitative interaction studies with a fluorescent peptide comprising the N-terminal -helix of HKI revealed a crucial role of the bilayer-facing glutamate in complex assembly and recapitulated an exquisite sensitivity of HKI-VDAC binding to fluctuations in pH. Our assay opens up important opportunities to investigate the impact of membrane environment on HKI-VDAC complex assembly and may benefit the development of therapeutics that target pathogenic imbalances in this process.

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Wessing, M., Watrinet, I., Timme, M., Fiedler, B., Melo, M. N., Piehler, J., Holthuis, J. C. M.. 2025-11-04. Key determinants of VDAC-hexokinase I complex assembly revealed by a minimal vesicle-based interaction assay. https://doi.org/10.1101/2025.11.03.686449

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