The serotonin 5-HT3 receptor is a pentameric ligand-gated ion channel (pLGIC). It belongs to a large family of receptors that function as allosteric signal transducers across the plasma membrane1,2: upon binding of neurotransmitter molecules to extracellular sites, the receptors undergo complex conformational transitions, which result in transient opening of a pore permeable to ions. 5-HT3 receptors are therapeutic targets for emesis and nausea, irritable bowel syndrome and depression3. Despite the recent accumulation in pLGIC structures4–8, no clear unifying view has emerged on conformational transitions involved in channel gating. Here we report four cryo-EM structures of the full-length mouse 5-HT3 receptor, ranging from 3.2 Å to 4.5 Å resolution, obtained in complex with the anti-emetic drug tropisetron, with serotonin, with serotonin and a positive allosteric modulator. The tropisetron-bound structure resembles those obtained with an inhibitory nanobody5 or without ligand9. The other structures represent new conformations, including that of an open state and of two novel ligand-bound states. We present computational insights into the dynamics of the structures, their pore hydration and free-energy profiles; we characterize motions at the gate level and cation accessibility in the pore. Put together, the data deepen our understanding of the gating mechanism of pLGICs, while capturing ligand binding in unprecedented detail.

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.