Biology subjects

Quentin, D.

Publications and source records attributed to Quentin, D..

5 recordsLinked to original sources

bioRxiv2021

Structure of a bacterial Rhs effector exported by the type VI secretion system

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 [A] resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise {beta}-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs cocoon where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal plug domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.

biochemistry↗

bioRxiv2021

Structure of the human FERRY Rab5 effector complex

Long-range mRNA transport is crucial for the spatio-temporal regulation of gene expression, and its malfunction leads to neurological disorders. The pentameric FERRY Rab5 effector complex is the molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of the human FERRY complex, composed of Fy-1 to Fy-5. The structure reveals a clamp-like architecture, where two arm-like appendages of Fy-2 and a Fy-5 dimer, protrude from the central Fy-4 dimer. The coiled-coil domains of Fy-2 are flexible and project into opposite directions from the core. While the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. The FERRY structure provides novel mechanistic insights into long-distance mRNA transport.

biochemistry↗

bioRxiv2020

Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones

Type VI secretion systems facilitate the delivery of antibacterial effector proteins between neighbouring Gram-negative bacteria. A subset of these effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across the target cell membrane. However, the abundance and distribution of these TMD-containing effectors has remained unknown. Here we report the discovery of prePAAR, a conserved motif found in over 6,000 putative TMD-containing effectors. Based on their differing sizes and number of TMDs these effectors fall into two distinct classes that are unified by their requirement for a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR functions to facilitate proper folding of the downstream PAAR domain, which is required for effector interaction with the T6SS spike. Taken together, our findings define the mechanism of chaperone-assisted secretion of a widespread family of T6SS membrane protein effectors.

biochemistry↗

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↗