Structural Models\nof Human Norepinephrine Transporter\nEnsemble Reveal the Allosteric Sites and Ligand-Binding Mechanism

The norepinephrine transporter (NET) plays a pivotal\nrole in recycling\nnorepinephrine (NE) from the synaptic cleft. However, the structures\nreferring to the conformational heterogeneity of NET during the transport\ncycle remain poorly understood. Here, three structural models of NE\nbound to the orthosteric site of NET in outward-open (OO<sub>holo</sub>), outward-occluded (OC<sub>holo</sub>), and inward-open (IO<sub>holo</sub>) conformations were first obtained using the multistate\nstructures of serotonin transporter as templates and further characterized\nthrough Gaussian-accelerated molecular dynamics and free energy reweighting.\nAnalysis of the structures revealed eight potential allosteric sites\non the functional-specific states of NET. One of the pharmacologically\nrelevant pockets located at the extracellular vestibule was further\nverified by simulating the binding behaviors of a clinical trial drug\nχ-MrIA that is allosterically regulating NET. These structural\nand energetic insights into NET advanced our understanding of NE reuptake\nand paved the way for discovering novel molecules targeting the allosteric\nsites.