Simultaneous control of composition and register of an AAB-type collagen heterotrimer.

Control over composition and register of the peptide chains in AAB-type collagen mimetic heterotrimers is critical in developing systems that show fidelity to native collagen. However, their design is challenging due to the eight competing states possible for a mixture of nonidentical peptides A and B. Interpeptide salt-bridges have been used previously as keystone interactions to bias the population of competing states to favor a target heterotrimer. The designed heterotrimers were electroneutral and relied on pairing of acidic and basic residues but could not differentiate between all of the competing states and reported systems populated either multiple heterotrimer compositions or registers. Here our design methodology includes both positive and negative elements. First, an excess of acidic or basic residues, which always remain unpaired, introduces a negative design component to destabilize the competing triple helical compositions and registers. Second, charge pairs introduce a positive design component and stabilize the target assembly. These antagonistic factors are optimized in the target heterotrimer that forms the maximum number of charge pairs and minimizes unpaired charged residues. Additionally, we find that not just the number of paired and unpaired residues are important, but also the type. By a systematic study of different types of charge pairs and unpaired residues, we are able to populate a single composition-single register AAB heterotrimer. The insights gained here may be useful in designing composition and register specific heterotrimeric ligands with domains that recognize collagen-binding proteins.