Binding and stability determinants of the PPARgamma 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 PPARgamma 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 PPARgamma was identified as necessary for PPARgamma-coactivator interaction. To decouple protein stability from the propensity to form a receptor-coactivator interface, libraries of PPARgamma 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 PPARgamma are also crucial for the stability of the PPARgamma.
Phillips
Kevin J
KJ
Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Rosenbaum
Daniel M
DM
Liu
David R
DR
D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D008958:Models, Molecular; D008969:Molecular Sequence Data; D047495:PPAR gamma / Q000378:metabolism*; D011485:Protein Binding
D047495:PPAR gamma; D000409:Alanine