Quentin, D. | Search bioRxiv

bioRxiv2020

Structural basis of TRPC4 regulation by calmodulin and pharmacological agents

ABSTRACTCanonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin. Molecular details of both calmodulin and drug binding have remained elusive so far. Here we report structures of TRPC4 in complex with a pyridazinone-based inhibitor and a pyridazinone-based activator and calmodulin. The structures reveal that both activator and inhibitor bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand binding site can be transmitted to the central ion-conducting pore of TRPC4. Calmodulin binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel calmodulin-induced regulatory mechanism of canonical TRP channels.Competing Interest StatementThe authors declare no competing interests. M.Y., M.W.L, and G.M. are or were shareholders of Goldfinch Bio.View Full Text

biochemistry↗

bioRxiv2020

Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state

Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called -clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the -clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the order of LF binding determines which factor is translocated first.

molecular biology↗