Cholesterol-dependent cytolysins (CDCs) are important virulence factors. An in-depth understanding of their membrane binding and pore formation mechanism is crucial for the development of their inhibitors and for their utilization in therapeutic, biotechnological, and synthetic biology applications.
The ribosome display technique enabled us to gain the protein binding scaffold based on designed ankyrin protein (DARPin) with a high binding affinity (KD ~ 100 nM) towards the Y406A, which is a pH-dependent mutant of CDC. Selected DARPin bound to the Y406A and inhibited its pore forming activity, although the membrane-binding step of Y406A was not affected. The DARPin was able to bind to the Y406A also when the Y406A was already inserted into a membrane. Implementation of ribosome display was successful also for selection of CDC membrane-binding domain variants (PFOD4) with different binding parameters. The wild-type PFOD4 bound to the membrane with 50 molar percent of cholesterol with a nanomolar affinity (KD). Between the two tested PFOD4 variants, one bound to the membrane cholesterol less specifically, namely to two differently accessible populations of membrane cholesterol, whereas both variants had the KDs in a similar range as the wild-type PFOD4.
Based on selective and specific inhibition of the CDC with the DARPin, we constructed the protein logic gate on a membrane and showed a proof of concept for such a system in synthetic biology. Ribosome display proved appropriate also for enrichment of membrane-binding domains with altered binding characteristics. Together with established techniques for measuring molecular interactions, ribosome display is an appreciated additional tool for studying protein-membrane interactions, with its main advantages being flexibility, allowance of numerous combinations of amino acid substitutions, and the possibility of affinity maturation.
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