Activity and Affinity of Pin1 Variants

Abstract

Pin1 is a peptidyl-prolyl isomerase responsible for isomerizing phosphorylated S/T-P motifs. Pin1 has two domains that each have a distinct ligand binding site, but only its PPIase domain has catalytic activity. Vast evidence supports interdomain allostery of Pin1, with binding of a ligand to its regulatory WW domain impacting activity in the PPIase domain. Many diverse studies have made mutations in Pin1 in order to elucidate interactions that are responsible for ligand binding, isomerase activity, and interdomain allostery. Here, we summarize these mutations and their impact on Pin1's structure and function.

Keywords: PPIase domain; WW domain; activity; affinity; mutants; pin1.

Figure 1

Structure of Pin1 and ligands. (A) Annotated crystal structure of PDB 1pin. (B) Primary pS/T-P peptide ligands discussed in this review.

Figure 2

Dissociation constants of Pin1 variants. K_D of (A) Pin1 variants including full-length (FL) and isolated domains to ligand pCDC25c, (B) mutants towards CTD RNA Pol II, (C) the WW domain and variants towards pCDC25c, and (D) full-length and isolated domains toward isomerizable and locked inhibitors of FFpSPR. Single measurements are reported only with a bar, while variants with multiple measurements show an error bar with the standard deviations. All values (with their individual experimental errors) are reported in the Supplemental Table S1. Variants with a large gray bar with an X had no detectable binding.

Figure 3

Activity of Pin1 Variants. Exchange rate K_EXSY of ligands (A) pCDC25c and (B) FFpSPR in presence of various Pin1 variants using exchange spectroscopy. Catalytic activity k_cat/K_M of Pin1 mutations using (C) chymotrypsin or (D) trypsin assay. Single measurements are reported only with a bar, while variants with multiple measurements show an error bar with the standard deviations. All values (with their individual experimental errors) are reported in the Supplemental Information. Variant R68A/R69A with an empty bar with an X had no detectable activity. The values reported in (D) are based on the estimate of WT Pin1 activity (~3200 mM⁻¹s⁻¹) from the original figure, and the estimates of percent of activity for each mutant compared to WT Pin1. Supplemental Table S3 also allows for 5% error in these values.

Figure 4

Overview of mutants that change affinity and activity of Pin1. Mutations that have been studied are plotted on the crystal structure 1pin. Mutations that negatively impact (A) affinity and (B) isomerase activity are labeled in red, positive mutations are in green, and neutral mutations are in yellow.