Highlights d CcdA antitoxin rejuvenates bacterial DNA gyrase by extracting bound CcdB toxin d CcdA forms transient ternary and quaternary complexes with gyrase:CcdB complex d Molecular basis for rejuvenation elucidated through computation and experiment d Similar methodology can be used to characterize other transient complexes

The majority of the proteins encoded in the genomes of eukaryotes contain more than one domain. Reasons for high prevalence of multi-domain proteins in various organisms have been attributed to higher stability and functional and folding advantages over single-domain proteins. Despite these advantages, many proteins are composed of only one domain while their homologous domains are part of multi-domain proteins. In the study presented here, differences in the properties of protein domains in single-domain and multi-domain systems and their influence on functions are discussed. We studied 20 pairs of identical protein domains, which were crystallized in two forms (a) tethered to other proteins domains and (b) tethered to fewer protein domains than (a) or not tethered to any protein domain. Results suggest that tethering of domains in multi-domain proteins influences the structural, dynamic and energetic properties of the constituent protein domains. 50% of the protein domain pairs show significant structural deviations while 90% of the protein domain pairs show differences in dynamics and 12% of the residues show differences in the energetics. To gain further insights on the influence of tethering on the function of the domains, 4 pairs of homologous protein domains, where one of them is a full-length single-domain protein and the other protein domain is a part of a multi-domain protein, were studied. Analyses showed that identical and structurally equivalent functional residues show differential dynamics in homologous protein domains; though comparable dynamics between in-silico generated chimera protein and multi-domain proteins were observed. From these observations, the differences observed in the functions of homologous proteins could be attributed to the presence of tethered domain. Overall, we conclude that tethered domains in multi-domain proteins not only provide stability or folding advantages but also influence pathways resulting in differences in function or regulatory properties.

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RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients or vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike mutants harbouring single and combinatorial amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing neutralisation. Moreover, introducing the 484 K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

Domain-domain interactions in multi-domain proteins play an important role in the combined function of individual domains for the overall biological activity of the protein. The functions of the tethered domains are often coupled and hence, limited numbers of domain architectures with defined folds are known in nature. Therefore, it is of interest to document the available fold-fold combinations and their preference in multi-domain proteins. Hence, we analyzed all multi-domain proteins with known structures in the protein databank and observed that only about 860 fold-fold combinations are present among them. Analyses of multi-domain proteins represented in sequence database result in recognition of 29,860 fold-fold combinations and it accounts for only 2.8% of the theoretically possible 1,036,080 (1439 C2) fold-fold combinations. The observed preference for fold-fold combinations in multi-domain proteins is interesting in the context of multiple functions through structural adaptation by gene fusion.

The accelerated development of the first generation COVID-19 vaccines has saved millions of lives, and potentially more from the long-term sequelae of SARS-CoV-2 infection. The most successful vaccine candidates have used the full-length SARS-CoV-2 spike protein as an immunogen. As expected of RNA viruses, new variants have evolved and quickly replaced the original wild-type SARS-CoV-2, leading to escape from natural infection or vaccine induced immunity provided by the original SARS-CoV-2 spike sequence. Next generation vaccines that confer specific and targeted immunity to broadly neutralising epitopes on the SARS-CoV-2 spike protein against different variants of concern (VOC) offer an advance on current booster shots of previously used vaccines. Here, we present a targeted approach to elicit antibodies that neutralise both the ancestral SARS-CoV-2, and the VOCs, by introducing a specific glycosylation site on a non-neutralising epitope of the RBD. The addition of a specific glycosylation site in the RBD based vaccine candidate focused the immune response towards other broadly neutralising epitopes on the RBD. We further observed enhanced cross-neutralisation and cross-binding using a DNA-MVA CR19 prime-boost regime, thus demonstrating the superiority of the glycan engineered RBD vaccine candidate across two platforms and a promising candidate as a broad variant booster vaccine.

Of the coronaviruses that have caused zoonotic spill overs in past two decades, the diverse group of beta-coronaviruses (β-CoVs) represent the greatest threats. Towards achieving broad vaccine protection from these viruses, vaccines composed of multiple antigens, each capable of eliciting broad neutralising responses across a subgroup will be required. Utilising a novel platform for selecting immune optimized, structurally engineered antigens capable of eliciting protective responses across a group of related viruses, we demonstrate proof-concept against the greater sarbecoviruses sub-genus with a single antigen structure. From an array of phylogenetically informed antigen structures displaying different broad neutralising epitopes, synthetic genes expressing these were selected based on broad immune responses in BALB/C mice. Improved protection against the Delta variant was further observed in K18-hACE2 mice on boosting with the lead designs of mice primed by an approved COVID-19 vaccine. Immunogenicity of the lead vaccine antigen and breadth of neutralisation against the SARS-CoV, SARS-CoV-2, WIV16, and RaTG13 was confirmed in guinea pigs using needleless intradermal immunisation. Immunogenicity was further confirmed in rabbits with GMP manufactured DNA immunogen. Notably, given the increasing number of mutations acquired by SARS-CoV-2 variants of concern (VOCs), the rabbit sera were tested for the capacity to neutralise VOCs - Beta, Gamma, Delta, as well as the most diverse Omicron variant. The consistent neutralising ability of the vaccine sera against the emerging VOCs validate broad specificity of the vaccine design. Here, we demonstrate proof-of-concept of this Digitally Immune Optimised, Selected vaccine (DIOSvax) antigen pipeline for the in vivo selection of single nucleic acid-based immunogens. Such gene-based antigens can be readily delivered alone or in combination, and seamlessly scaled with vaccine delivery modalities such as viral vector or mRNA-based vaccines.