Hypoxia-driven phosphatidylserine remodeling promotes conformational plasticity of ADAM17 in head and neck squamous cell carcinoma

Introduction. A Disintegrin and Metalloprotease 17 (ADAM17) is a critical regulator of cancer progression through the shedding of growth factors, cytokines, and adhesion molecules. In head and neck squamous cell carcinoma (HNSCC), aggressive disease is closely associated with hypoxic tumor regions that drive adaptive signaling, extracellular communication, and therapy resistance. In our previous work, we demonstrated that hypoxia enhances the release of small extracellular vesicles (sEVs) from HNSCC cells and that ADAM17 is actively carried by these vesicles. Notably, sEVs derived under hypoxic conditions exhibited higher ADAM17 activity compared with normoxic sEVs, an effect significantly attenuated by phosphatidylserine (PS) antibody. Due to these observations, we hypothesized that ADAM17 assumes different conformational states with different levels of activity, and that PS engagement triggers a change towards more active states. Methods. To investigate lipid-mediated regulation of ADAM17, we performed molecular dynamics simulations and trajectory analysis (GROMACS, PACKMOL, CHARMM36, CGenFF) using membrane models (20nm 2 ) bulid on PS composition informed by lipidomics analysis of sEVs derived from hypoxic and normoxic HNSCC cell line (PCI-30). Mass spectrometry lipidomic profiling revealed distinct PS species differing in acyl chain length and saturation between conditions. Based on these data, we constructed membrane systems reflecting the experimentally observed PS compositions. A structural model of ADAM17 (PDB 8SNM) was embedded in these membranes, and timescale simulations (5 ns) were conducted to assess lipid-protein interactions, PS binding dynamics, and conformational responses of ADAM17 under hypoxia- and normoxia-mimicking conditions, producing free energy landscapes and transition rates between metastable states. Results. Molecular dynamics simulations revealed that ADAM17 samples distinct conformational ensembles in membranes reflecting hypoxic versus normoxic tumor conditions, driven by differences in phosphatidylserine (PS) acyl chain length and saturation identified by sEV lipidomics. These hypoxic PS compositions stabilized open-like ADAM17 conformations, characterized by increased membrane-proximal domain tilt angles (≈92°, mean Δ = 3.1°), increased backbone RMSD fluctuations (ΔRMSD ≈ −0.79 Å), and increased separation between the membrane surface and the catalytic domain (Δdistance ≈ +25 Å) relative to normoxic membranes. Free-energy analysis revealed a population shift toward lower-energy open states under hypoxic PS conditions (ΔG ≈ −1.0 kJ/mol), providing a molecular mechanism linking tumor hypoxia-driven lipid remodeling to enhanced ADAM17 activity. Conclusion. Our results provide a molecular framework linking hypoxia-driven lipid remodeling of sEV membranes to enhanced ADAM17 activity in HNSCC. By integrating lipidomics-guided membrane modeling with molecular simulations, this study highlights phosphatidylserine as a key regulator of ADAM17 function in aggressive tumors. Targeting PS-dependent lipid-protein interactions may represent a novel strategy to modulate ADAM17-mediated signaling in hypoxic cancer microenvironments.