A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site
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The prolyl isomerase Pin1 catalyzes the cis-trans isomerization 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 proline cis-trans isomerization of pS/pT-P motifs. Here, we show that Pin1 uses a tethering mechanism to bind and catalyze proline cis-trans isomerization 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 accelerates cis-trans isomerization 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 of cis-trans isomerization 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 catalytic cis-trans isomerization at a distal noncanonical site.
SIGNIFICANCE
Proline peptide bonds naturally occur in cis conformations and isomerize to trans conformations 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.
