Transmembrane Domain Oligomerization and Intracellular Domain–Lipid Interaction Oppositely Modulates OX40 Receptor Activation Unveiled by ¹⁹ F NMR

The OX40 (CD134) receptor is an important drug target for antitumor and autoimmune therapies, yet its signaling mechanisms remain poorly understood. Here, we present the first investigation of the OX40 transmembrane domain (TMD) and intracellular domain (ICD), as well as their association with downstream TRAF2, within near-native membrane environments using a combination of chemical and biophysical methods. Using fluorine nuclear magnetic resonance (19F NMR), double electron–electron resonance (DEER), and isothermal titration calorimetry (ITC), we demonstrate that the association of downstream TRAF2 with the acid-rich sequence (ARS) signaling motif of OX40 is oppositely modulated by the TMD oligomerization and ICD–lipid interaction. Specifically, receptor clustering mediated by TMD oligomerization enhances TRAF2 association, whereas ICD–lipid interaction inhibits it. This work reveals a novel regulatory mechanism for OX40, suggesting that its signal transduction and activation are governed by a dynamic tug-of-war between lipid–ICD interaction-mediated inhibition and TMD oligomerization-driven activation.