Distinct Effects of the Extracellular Domain and Antibody Binding on HER2 Transmembrane and Juxtamembrane Regions Unveiled by NMR and EPR

The HER2 receptor, a critical member of the human epidermal growth factor receptor (EGFR) family, plays a crucial role in cancer progression. Its transmembrane region, containing the extracellular juxtamembrane (EJM) region, transmembrane domain (TMD), and intracellular juxtamembrane (IJM) region, undergoes complex conformational dynamics that are essential for its functional activities. However, these dynamic properties within lipid bilayers remain poorly understood. This study employs a combination of fluorine nuclear magnetic resonance ( 19 F NMR), pulsed dipolar electron paramagnetic resonance (PD-EPR), and a nanodisc strategy with a membrane-proximal chemical reporter (NMPCR) to elucidate HER2 EJM-TMD-IJM’s conformational changes induced by extracellular domain (ECD) incorporation and therapeutic single-domain antibody (sdAb) binding in a near-native membrane environment. Our results demonstrate that the ECD incorporation greatly restricts the conformation flexibility or heterogeneity of both TMD and IJM in HER2. Furthermore, binding of sdAb to HER2 ECD introduces large conformational rearrangements of the EJM region. These insights enhance our understanding of HER2’s molecular behavior within lipid bilayers, provide significant implications for targeted cancer therapies, and establish a generalizable workflow for studying transmembrane dynamics in near-native lipid environments.