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Abstract: Protein translocation across the endoplasmic reticulum (ER) membrane is an essential step during protein entry into the secretory pathway. The conserved Sec61 protein-conducting channel facilitates polypeptide translocation and coordinates cotranslational polypeptide-processing events. In cells, the majority of Sec61 is stably associated with a heterotetrameric membrane protein complex, the translocon-associated protein complex (TRAP), yet the mechanism by which TRAP assists in polypeptide translocation remains unknown. Here, we present the structure of the core Sec61/TRAP complex bound to a mammalian ribosome by cryogenic electron microscopy (cryo-EM). Ribosome interactions anchor the Sec61/TRAP complex in a conformation that renders the ER membrane locally thinner by significantly curving its lumenal leaflet. We propose that TRAP stabilizes the ribosome exit tunnel to assist nascent polypeptide insertion through Sec61 and provides a ratcheting mechanism into the ER lumen mediated by direct polypeptide interactions.

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Abstract: Carboxysomes are a paradigm of self-assembling proteinaceous organelles found in nature, offering compartmentalisation of enzymes and pathways to enhance carbon fixation. In α-carboxysomes, the disordered linker protein CsoS2 plays an essential role in carboxysome assembly and Rubisco encapsulation. Its mechanism of action, however, is not fully understood. Here we synthetically engineer α-carboxysome shells using minimal shell components and determine cryoEM structures of these to decipher the principle of shell assembly and encapsulation. The structures reveal that the intrinsically disordered CsoS2 C-terminus is well-structured and acts as a universal “molecular thread” stitching through multiple shell protein interfaces. We further uncover in CsoS2 a highly conserved repetitive key interaction motif, [IV]TG, which is critical to the shell assembly and architecture. Our study provides a general mechanism for the CsoS2-governed carboxysome shell assembly and cargo encapsulation and further advances synthetic engineering of carboxysomes for diverse biotechnological applications.

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Abstract: The human endogenous retrovirus K (HERV-K) is the most recently acquired endogenous retrovirus in the human genome and is activated and expressed in many cancers and amyotrophic lateral sclerosis. We present the immature HERV-K capsid structure at 3.2 Å resolution determined from native virus-like particles using cryo-electron tomography and subtomogram averaging. The structure shows a hexamer unit oligomerized through a 6-helix bundle, which is stabilized by a small molecule analogous to IP6 in immature HIV-1 capsid. The HERV-K immature lattice is assembled via highly conserved dimer and trimer interfaces, as detailed through all-atom molecular dynamics simulations and supported by mutational studies. A large conformational change mediated by the linker between the N-terminal and the C-terminal domains of CA occurs during HERV-K maturation. Comparison between HERV-K and other retroviral immature capsid structures reveals a highly conserved mechanism for the assembly and maturation of retroviruses across genera and evolutionary time.

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Abstract: Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4–Not deadenylation complex. The details of this process in mammals remain unclear. Here, we use cryogenic electron microscopy (cryo-EM) and crosslinking mass spectrometry to show that mammalian CCR4–NOT specifically recognizes ribosomes that are stalled during translation elongation in an in vitro reconstituted system with rabbit and human components. Similar to yeast, mammalian CCR4–NOT inserts a helical bundle of its CNOT3 subunit into the empty E site of the ribosome. Our cryo-EM structure shows that CNOT3 also locks the L1 stalk in an open conformation to inhibit further translation. CCR4–NOT is required for stable association of the nonconstitutive subunit CNOT4, which ubiquitinates the ribosome, likely to signal stalled translation elongation. Overall, our work shows that human CCR4–NOT not only detects but also enforces ribosomal stalling to couple translation and mRNA decay.

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Abstract: Membranes confer cells with individual identity and capacity to regulate their response to their environment. A critical aspect of having a membranous partition is the ability to transport substances into and out of cells as part of life-sustaining functions. In pathogenic bacteria, transporters aid infection and survival in the host. Two such transporters in Gram- negative bacterial species are the MacA-MacB-TolC (MacAB-TolC) antibiotic efflux pump and the Type I Secretion System (T1SS), responsible respectively for antibiotic resistance and export of protein virulence factors. To pass the Gram-negative envelope in a one-step translocation process, both machines use a tripartite system, consisting of outer membrane protein TolC, a periplasmic adapter protein (MacA or haemolysin D (HlyD) in the T1SS), and an inner membrane protein (MacB or haemolysin B (HlyB) in the T1SS). Both use the power of ATP-hydrolysis to export their substrates. Here, I utilise computational and experimental approaches to elucidate the mechanism of function for both machines. I conduct molecular dynamics (MD) simulations of membrane embedded HlyB component of the T1SS with and without its haemolysin A (HlyA) substrate as in silico experiments. I also conduct MD simulations with and without substrate for a related peptidase. I show that substrate recognition is via conserved charge-charge interactions. I also show that HlyB has an asymmetric preferential interaction with cardiolipin when its substrate is present, which is not seen in the peptidase simulations. I propose that this preference is part of the mechanism of transport, with cardiolipin providing energy via the proton-motive force. I test this hypothesis through flow cytometry detection of labelled substrate trapped T1SS in a mixed population of cells, by comparing parental MG1655 Escherichia coli with a cardiolipin deficient MG1655 strain. I found that the cardiolipin deficient strain has reduced T1SS levels compared to its parent. To aid structural studies, I optimise the expression of the T1SS using a flow cytometry based sequential design strategy where conditions are iteratively tested via detection of substrate trapped T1SS and updated until no more improvement can be made. I also test purification strategies for single-particle cryo-electron microscopy studies. Finally, I apply further bioinformatic approaches and synthesise my computational and experimental results to propose a mechanism of transport and suggest future experimental tests. I conduct MD simulations of MacB in membrane with and without a trapped lipid. I show that this trapped lipid locks MacB into an open state, allowing for substrate entry into the pump. I contextualise the results by comparing MD simulations to MacB-like structures and propose a revised mechanism of transport as a function of its free-energy landscape. Lastly, I explore the use of cryo-electron tomography (cryo-ET) as a method to obtain in vivo structural insights. I show that the use of “ghost” partially lysed E. coli can produce high-contrast specimens for tomography. I collect a tomographic dataset of “ghost” MacAB-TolC containing cells and apply subtomogram averaging. Preliminary results suggest that MacAB-TolC forms an array in cells, and that MacB is structurally flexible, likely in its nucleotide-binding domain. Together, these studies of the MacAB-TolC efflux pump and the T1SS shed light on their function and suggest new avenues of research to explore in order to fulfil the goal of finding novel inhibitors.