Interactions with lipid membrane modulate the conformational dynamics and energy landscape of Tumor Necrosis Factor

Tumor Necrosis Factor (TNF) is a trimeric cytokine that exists in soluble (sTNF) and membrane-bound (mTNF) forms, both of which regulate immune responses through interactions with their cognate receptors. sTNF has been the subject of extensive biophysical investigations, leading to a mechanistic model in which a symmetric arrangement of the trimer promotes receptor signaling, whereas asymmetric conformations inhibit this function. In contrast, the structural and energetic landscape of mTNF remains largely under-explored. Here, we combined multi-microsecond unbiased molecular dynamics and Metadynamics-Adaptive Biasing Force simulations to characterize mTNF embedded in a physiologically relevant lipid bilayer. We show that in the absence of membrane engagement, the extracellular domain (ECD) dynamically samples multiple asymmetric conformations similar to those observed in sTNF. The association of the ECD with the membrane, mediated primarily by the basic residues R78-R82, R107, R108, R120, and R207, restricts conformational heterogeneity and stabilizes the symmetric state. By quantifying the energetic effects of ECD-membrane association, we demonstrate that the symmetric state in the membrane-bound ECD is stabilized over asymmetric conformations to a greater extent than in sTNF. This energetic effect, together with the spatial confinement imposed by the lipid bilayer, may explain the reported reduced affinity of certain biologics for mTNF. In conclusion, our work elucidates how the membrane influences the structure, dynamics, and energetics of TNF. These mechanistic insights could guide future efforts to design mTNF-selective inhibitors that account for both membrane constraints and the energetic modulation of the ECD conformational landscape.