A tethering mechanism underlies Pin1-catalyzed prolinecis-transisomerization at a noncanonical site

Abstract

ABSTRACTThe prolyl isomerase Pin1 catalyzes thecis-transisomerization of proline peptide bonds, a non-covalent post-translational modification that influences cellular and molecular processes, including protein-protein interactions. Pin1 is a two-domain enzyme containing a WW domain that recognizes phosphorylated serine/threonine-proline (pS/pT-P) canonical motifs and an enzymatic PPIase domain that catalyzes prolinecis-transisomerization of pS/pT-P motifs. Here, we show that Pin1 uses a tethering mechanism to bind and catalyze prolinecis-transisomerization of a noncanonical motif in the disordered N-terminal activation function-1 (AF-1) domain of the human nuclear receptor PPARγ. NMR reveals multiple Pin1 binding regions within the PPARγ AF-1, including a canonical motif that when phosphorylated by the kinase ERK2 (pS112-P113) binds the Pin1 WW domain with high affinity. NMR methods reveal that Pin1 also binds and acceleratescis-transisomerization of a noncanonical motif containing a tryptophan-proline motif (W39-P40) previously shown to be involved in an interdomain interaction with the C-terminal ligand-binding domain (LBD). Cellular transcription studies combined with mutagenesis and Pin1 inhibitor treatment reveal a functional role for Pin1-mediated acceleration ofcis-transisomerization of the W39-P40 motif. Our data inform a refined model of the Pin1 catalytic mechanism where the WW domain binds a canonical pS/T-P motif and tethers Pin1 to the target, which enables the PPIase domain to exert catalyticcis-transisomerization at a distal noncanonical site.SIGNIFICANCEProline peptide bonds naturally occur incisconformations and isomerize totransconformations on exchange regimes on the order of seconds to minutes. Pin1, a prolyl isomerase, catalyzes the isomerization of proline peptide bonds that contain a specific phospho-motif—a phosphorylated serine or threonine followed by a proline (pS/pT-P)—allowing for switch-like effects on target protein structure and function. One protein substrate of Pin1 is the nuclear receptor peroxisome proliferator activated receptor gamma (PPARγ), which is shown here to undergo Pin1-catalyzed isomerization at a noncanonical proline distal to a canonical pS/pT-P binding site. These studies lay the foundation for understanding the role of Pin1 in mediating PPARγ-regulated transcription and expand understanding of Pin1-catalyzed enzymatic activities and functions.