Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site.

doi: 10.1073/pnas.2414606122

Figure Lengend Snippet: Fig. 2. Pin1 binding to the AF-1 is enhanced by ERK2 phosphorylation. Overlays of 2D [1H,15N]-HSQC NMR spectra of (A) 15N-labeled AF-1, (B) 15N-labeled pAF-1, and (C) 15N-labeled pAF-1 P76A mutant without or with 1 molar equivalent of human Pin1 reveal larger chemical shift perturbations (CSPs) in the phosphorylated state indicating stronger binding of Pin1 to phosphorylated AF-1. Peaks belonging to regions of particular interest noted by blue and green labeling are discussed further in the results. (D) AlphaFold3 model of Pin1 interaction with pAF-1 containing phosphorylated S112 (pS112) and T75 (pT75) residues with NMR CSPs from 15N-labeled pAF-1 ± 1x Pin1 (2B) shows the Pin1-binding epitope on pAF-1 extends to regions beyond the pS112 region. Spheres represent residue alpha carbons colored by CSP magnitude [white (CSP = 0) to magenta (CSP = 0.03)] or noting peaks that disappear (black). CSP plots comparing addition of Pin1 full-length, WW domain, or PPIase domain at 1 molar equivalent to (E) 15N-labeled AF-1, (F) 15N-labeled pAF-1, and (G) 15N-labeled pAF-1 P76A mutant reveal that each Pin1 domain binds weakly to AF-1, but binding of full-length Pin1 elicits larger CSPs in targeted regions of pAF-1 and pAF-1 P76A mutant.

Article Snippet: Full- length human Pin1 and the Pin1 WW domain (1–39) protein were expressed from a pMCSG7 plasmid vector, purchased through Addgene (#40773) as TEV- cleavable N- terminal 6xHis fusion proteins.

Techniques: Binding Assay, Phospho-proteomics, Labeling, Mutagenesis, Residue

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site.

doi: 10.1073/pnas.2414606122

Figure Lengend Snippet: Fig. 4. AF-1 peptides delineating contributions of specific AF-1 regions in binding to Pin1. (A) Sequence and relative locations of peptides corresponding to AF-1 regions that include the S112 phosphorylation site with unphosphorylated S112 (green) or phosphorylated S112 (teal); T75 phosphorylation site with unphosphorylated T75 (purple) or phosphorylated T75 (coral); and the PFWP motif (mustard). Overlays of 2D [1H,15N]-TROSY HSQC NMR spectra of 15N-labeled Pin1 with (B) S112, (C) T75, (D) pS112, (E) pT75, and (F) W39 peptides titrated up to 5 molar equivalents. Shown alongside spectra are AlphaFold3 models of Pin1 colored by magnitude of CSP [pale blue/white (CSP = 0) to magenta (CSP = 0.1)] elicited by 2X titration of respective peptide shown in complex with Pin1. The WW domain is colored pale blue, and the PPIase domain is white. Peptides in models are colored corresponding to their identity as indicated in 5A. (G) CSP plots comparing addition of unphosphorylated or phosphorylated peptides at 5 molar equivalents to 15N-labeled Pin1. CSP profiles reveal Pin1 interaction patterns unique to each AF-1 peptide, with phosphorylated AF-1 peptides driving enhanced interaction to the WW domain.

Article Snippet: Full- length human Pin1 and the Pin1 WW domain (1–39) protein were expressed from a pMCSG7 plasmid vector, purchased through Addgene (#40773) as TEV- cleavable N- terminal 6xHis fusion proteins.

Techniques: Binding Assay, Sequencing, Phospho-proteomics, Labeling, Titration

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site.

doi: 10.1073/pnas.2414606122

Figure Lengend Snippet: Fig. 6. Approaches to studying Pin1 binding and catalysis. Previous structural studies of Pin1 isomerization depended heavily on the use of short peptide sequences (~20 amino acids). In addition to short peptides, our studies using a larger protein domain with multiple potential canonical and noncanonical Pin1 sites uncovered a multivalent binding interaction that catalyzes a noncanonical W-P motif with functional cellular relevance.

Article Snippet: Full- length human Pin1 and the Pin1 WW domain (1–39) protein were expressed from a pMCSG7 plasmid vector, purchased through Addgene (#40773) as TEV- cleavable N- terminal 6xHis fusion proteins.

Techniques: Binding Assay, Functional Assay