Designed Ankyrin Repeat Proteins provide insights into the structure and function of CagI and are potent inhibitors of CagA translocation by theHelicobacter pyloritype IV secretion system

AbstractThe bacterial human pathogenHelicobacter pyloriproduces a type IV secretion system (cagT4SS) to inject the oncoprotein CagA into gastric cells. ThecagT4SS external pilus mediates attachment of the apparatus to the target cell and the delivery of CagA. While the composition of the pilus is unclear, CagI is present at the surface of the bacterium and required for pilus formation. Here, we have investigated the properties of CagI by an integrative structural biology approach. Using Alpha Fold 2 and Small Angle X-ray scattering, it was found that CagI forms elongated dimers mediated by rod-shaped N-terminal domains (CagIN) and prolonged by globular C-terminal domains (CagIC). Three Designed Ankyrin Repeat Proteins (DARPins) K2, K5 and K8 selected against CagI interacted with CagICwith subnanomolar affinities. The crystal structures of the CagI:K2 and CagI:K5 complexes were solved and identified the interfaces between the molecules, thereby providing a structural explanation for the difference in affinity between the two binders. Purified CagI and CagICwere found to interact with adenocarcinoma gastric (AGS) cells, induced cell spreading and the interaction was inhibited by K2. The same DARPin inhibited CagA translocation by up to 65% in AGS cells while inhibition levels were 40% and 30% with K8 and K5, respectively. Our study suggests that CagICplays a key role incagT4SS-mediated CagA translocation and that DARPins targeting CagI represent potent inhibitors of thecagT4SS, a crucial risk factor for gastric cancer development.Author summaryHelicobacter pyloriis a bacterial pathogen that colonises the human stomach in half of the world’s population. The most virulent strains use the cag- type IV secretion system (cagT4SS), a molecular nanomachine capable of injecting the oncoprotein CagA into gastric cells. How CagA is delivered is unknown, but thecagT4SS produces an external appendage referred to as pilus, which interacts with host cell receptors, mediating CagA translocation from the cytoplasm of the bacteria to the inner membrane of the host cell. In this study we have investigated the structural and functional properties of CagI, a protein long-thought to be associated with thecagT4SS pilus but with yet unknown function. We found that CagI displays a unique dimeric structure and that its C-terminal domain is involved in interaction with the host cell. Designed Ankyrin Repeat Proteins were selected against CagI and found to interact with its C-terminal moiety with high affinity. DARPin binding was able to prevent CagI interaction with the host cell and inhibited CagA translocation byH. pylori. Our study reveals the role of CagI incagT4SS interaction with gastric cells and provides a first example of a small protein binder inhibiting thecagT4SS activity.