Pin1 is a peptidyl-prolyl isomerase consisting of a WW domain and a catalytic isomerase (PPIase) domain connected by a flexible linker. Pin1 recognizes phospho-Ser/Thr-Pro motifs in cell-signaling proteins, and is both a cancer and an Alzheimer's disease target. Here, we provide novel insight into the functional motions underlying Pin1 substrate interaction using nuclear magnetic resonance deuterium ((2)D) and carbon ((13)C) spin relaxation. Specifically, we compare Pin1 side-chain motions in the presence and absence of a known phosphopeptide substrate derived from the mitotic phosphatase Cdc25. Substrate interaction alters Pin1 side-chain motions on both the microsecond-millisecond (mus-ms) and picosecond-nanosecond (ps-ns) timescales. Alterations include loss of ps-ns flexibility along an internal conduit of hydrophobic residues connecting the catalytic site with the interdomain interface. These residues are conserved among Pin1 homologs; hence, their dynamics are likely important for the Pin1 mechanism.

The current canon attributes the binding specificity of protein-recognition motifs to distinctive chemical moieties in their constituent amino acid sequences. However, we show for a WW domain that the sequence crucial for specificity is an intrinsically flexible loop that partially rigidifies upon ligand docking. A single-residue deletion in this loop simultaneously reduces loop flexibility and ligand binding affinity. These results suggest that sequences of recognition motifs may reflect natural selection of not only chemical properties but also dynamic modes that augment specificity.

Summary Pin1 is a modular peptidyl-prolyl isomerase specific for phosphorylated Ser/Thr-Pro (pS/T-P) motifs, typically within intrinsically disordered regions (IDRs) of signaling proteins. Pin1 consists of two flexibly linked domains: an N-terminal WW domain for substrate binding and a larger C-terminal peptidyl-prolyl isomerase (PPIase) domain. Previous studies showed that binding of phosphopeptide substrates to Pin1 could alter Pin1 interdomain contact, strengthening or weakening it, depending on the substrate sequence. Thus, substrate-induced changes in interdomain contact may act as a trigger within the Pin1 mechanism. Here, we investigate this possibility via NMR studies of several Pin1 mutants. Our findings provide new mechanistic insights for those substrates that reduce interdomain contact. Specifically, the reduced interdomain contact can allosterically enhance PPIase activity relative to that when the contact is sustained. These findings suggest Pin1 interdomain contact can negatively regulate its activity.

Protein-protein interactions are often mediated by flexible loops that experience conformational dynamics on the microsecond to millisecond time scales. NMR relaxation studies can map these dynamics. However, defining the network of inter-converting conformers that underlie the relaxation data remains generally challenging. Here, we combine NMR relaxation experiments with simulation to visualize networks of inter-converting conformers. We demonstrate our approach with the apo Pin1-WW domain, for which NMR has revealed conformational dynamics of a flexible loop in the millisecond range. We sample and cluster the free energy landscape using Markov State Models (MSM) with major and minor exchange states with high correlation with the NMR relaxation data and low NOE violations. These MSM are hierarchical ensembles of slowly interconverting, metastable macrostates and rapidly interconverting microstates. We found a low population state that consists primarily of holo-like conformations and is a “hub” visited by most pathways between macrostates. These results suggest that conformational equilibria between holo-like and alternative conformers pre-exist in the intrinsic dynamics of apo Pin1-WW. Analysis using MutInf, a mutual information method for quantifying correlated motions, reveals that WW dynamics not only play a role in substrate recognition, but also may help couple the substrate binding site on the WW domain to the one on the catalytic domain. Our work represents an important step towards building networks of inter-converting conformational states and is generally applicable.

Conformational dynamics are drawing increasing attention in drug discovery due to their effect on the stability and diversity of protein-ligand complexes. 1 Both protein and ligand dynamics are important. Yet, the ligand has received less scrutiny, despite being the object of iterative modification in drug design. Accordingly, we apply here 13 C Nuclear Magnetic Resonance (NMR) relaxation dispersion measurements, at natural abundance, to a ligand in chemical exchange between free and protein-bound states. Specifically, we compare site-specific relaxation dispersion profiles of ligand 13 C nuclei in the presence and absence of its protein receptor. Profiles that differ are 13 C sites whose chemical shifts change upon protein binding. Fits of the profiles give the rate constants for the exchange of ligand between its free versus bound conformations. Because these experiments detect the ligand, they are applicable even in the absence of a protein structure. While previous ligand mobility studies have used natural abundance 13 C relaxation, 2-6 this study, to the best of our knowledge, is the first demonstration of 13 C relaxation dispersion at natural abundance on a ligand in protein-binding exchange.Peptide-protein interactions of modest binding affinity often guide the early stages of drug design, 7 and we illustrate the dispersion methods on a 10-residue phosphopeptide that binds human Pin1 with K D ~10 μM. 8 Pin1 is a 163 residue prolyl isomerase that recognizes phosphoSer/Thr-Pro (pS/T-P) motifs in other signaling proteins relevant for cancer and Alzheimer's disease. 9 Our ligand (EQPLpTPVTDL) contains a Pin1 pT-P target site from the mitotic phosphatase, Cdc25, a Pin1 substrate.Chemical exchange of ligands between their free and receptorbound states can modulate the ligand chemical shifts, thus boosting the ligand R 2 = 1/T 2 relaxation rates by an amount R ex . 10,11 Twodimensional (2D) relaxation dispersion measurements profile R ex versus spin-lock field strength in a site-specific manner. 12 Fits of the profiles yield the exchange rates and, under favorable exchange regimes, the underlying chemical shift differences and populations of the exchange coupled states. 13We explored 13 C relaxation dispersion since it is now widely accepted that 13 Cα, β chemical shifts reflect primarily backbone φ, ψ, and side chain χ 1 torsion angles. 14-20 Those 13 Cα sites showing relaxation dispersion, and, hence, chemical shift modulation, are candidate sites of φ, ψ, and χ 1 fluctuations on the μs-ms time scale. This dynamic interpretation of 13 Cα relaxation dispersion has been used in studies of 13 C-labeled proteins. [21][22][23] Correspondence to: Jeffrey W. Peng. E-mail: jpeng@nd.edu. Supporting Information Available: NMR spectra, parameter fitting procedures, relaxation rates for all residues. The material is available free of charge via the Internet at http://pubs.acs.org. While 13 C labeling is relatively facile for proteins, it is often impractical for ligands, in pharmaceutical research settings, due to cost or...