Double barrel shotgun scanning of the caveolin-1 scaffolding domain

In the postgenomic era, a major challenge remains, elucidating the thermodynamic forces governing receptorligand specificity and promiscuity. We report a straightforward approach for mapping side-chain contributions to binding for the multipartner interactions characteristic of the human proteome. Double barrel shotgun scanning dissects binding to two or more targets through combinatorial mutagenesis of one protein binding to multiple targets. Examined here, the caveolin-1 scaffolding domain (CSD) binds to and inhibits both endothelial nitric oxide synthase (eNOS) and protein kinase A (PKA). Homolog shotgun scanning of CSD highlights residues responsible for CSD oligomerization and binding to eNOS and PKA. The experiments uncover a general mechanism in which CSD oligomerizes and deoligomerizes to modulate binding affinity to partner proteins. The results provide a detailed look at a multipartner protein interaction, uncovering strategies for one protein binding to multiple partners.