Binding and stability determinants of the PPARγ nuclear receptor-coactivator interface as revealed by shotgun alanine scanning and in vivo selection

We modified an existing selection for protein-protein interactions based on the fragment complementation of the enzyme DHFR. Using shotgun alanine scanning in conjunction with this selection, we analyzed the interaction of the nuclear receptor PPARγ with two peptides derived from nuclear receptor coactivators SRC1 and TRAP220. A large binding epitope stretching between and including the charge clamp residues K301 and E471 of PPARγ was identified as necessary for PPARγ-coactivator interaction. To decouple protein stability from the propensity to form a receptor-coactivator interface, libraries of PPARγ variants generated by shotgun scanning were further processed using a high-throughput screen measuring their in vivo stabilities. Our findings demonstrate that many of the residues that make up the binding epitope of PPARγ are also crucial for the stability of the PPARγ.