Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the “5 + 5 inverted repeat” fold found in other families like the Na + -coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg + -bound occluded. However, the role of Arg + during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg + -bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg + -guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.

Objectives Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion‐stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 ‘MF59C.1’ (Seqirus, Parkville, Australia). Methods A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo‐electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. Results In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S‐specific CD4 + and cytotoxic CD8 + T cells in vivo . In the Syrian hamster challenge model ( n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. Conclusion The SARS‐CoV‐2 Sclamp vaccine candidate is compatible with large‐scale commercial manufacture, stable at 2–8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T‐cell responses and provides protection in animal challenge models.

SummaryThe minimal replication origin of the broad-hostrange plasmid RK2, oriV , contains five iterons which are binding sites for the plasmid-encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT-rich region containing four 13-mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13-mer motifs have been constructed and analysed for replication activity in vivo and in vitro . The data show that the replacement of oriV 13-mers by similar but not identical 13-mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13-mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of singlenucleotide substitutions demonstrates very restricted sequence requirements depending on the 13-mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli . Our experiments demonstrate considerable complexity in the plasmid AT-rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence-specific protein interactions within the 13-mers during assembly of replication complexes at the plasmid origin.

Prokaryotic and eukaryotic replicons possess a distinctive region containing a higher than average number of adenine and thymine residues (the AT-rich region) where, during the process of replication initiation, the initial destabilization (opening) of the double helix takes place. In many prokaryotic origins, this region consists of repeated 13-mer motifs whose function has not yet been specified. Here we identify specific mutations within the 13-mer sequences of the broad-host-range plasmid RK2 that can result in defective origin opening or that do not affect opening but induce defects in helicase loading. We also show that after the initial recruitment of helicase at the DnaA-box sequences of the plasmid origin, the helicase is translocated to the AT-rich region in a reaction requiring specific sequence of the 13-mers and appropriate facing of the origin motifs. Our results demonstrate that specific sequences within the AT-rich region of a replication origin are required for either origin opening or helicase loading.13-mer sequences ͉ DNA replication ͉ DnaB helicase ͉ plasmid RK2 T he initiation of DNA synthesis requires a mechanism to coordinate DNA unwinding, helicase recruitment, and sitespecific helicase loading. Binding of the initiator (Rep), a single protein, or a complex of several proteins to the replication origin results in the melting of the double-stranded DNA helix in a region of the origin with low internal thermodynamic stability because of a higher than average content of adenine and thymine residues (AT-rich region). In plasmid replicons, the involvement of both the host-encoded replication initiator DnaA and the plasmid encoded Rep protein is essential for origin melting, helicase recruitment, and helicase loading onto the ssDNA in the opened AT-rich region.DNA replication of the broad-host-range plasmid RK2 is initiated by the binding of the plasmid-encoded protein TrfA to direct repeats (iterons) present at its replication origin (oriV), a process similar to that of other plasmid systems (1, 2). The formation of an open complex at oriV by the TrfA initiation protein requires HU and is stabilized by the host DnaA protein (3). In contrast, the initiation of replication at the Escherichia coli chromosomal origin, oriC, involves a single initiator, DnaA, binding to DnaA-box sequences and resulting in duplex DNA melting (4-7). During replication initiation at RK2 oriV, helix destabilization occurs within the AT-rich region, which includes four 13-mer repeated sequences (L, M1, M2, R) that exhibit a DNA consensus sequence of TAAACnTTnTTTT (3). Similar motifs have been identified in a majority of theta-replicating plasmids and bacterial chromosomes including E. coli, where three 13-mers (L, M and R) have been identified in oriC (4-6). The position of the 13-mer clusters and their specific sequences are important for proper functioning of both oriV (8, 9) and oriC (5, 10, 11). Although the importance of 13-mers is well established, their exact role is not completely understood.Certain E. coli pro...

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