Extended Impact of Pin1 Catalytic Loop Phosphorylation Revealed by S71E Phosphomimetic.

Pin1 is a two-domain human protein that catalyzes the cis-trans isomerization of phospho-Ser/Thr-Pro (pS/T-P) motifs in numerous cell-cycle regulatory proteins. These pS/T-P motifs bind to Pin1's peptidyl-prolyl isomerase (PPIase) domain in a catalytic pocket, between an extended catalytic loop and the PPIase domain core. Previous studies showed that post-translational phosphorylation of S71 in the catalytic loop decreases substrate binding affinity and isomerase activity. To define the origins for these effects, we investigated a phosphomimetic Pin1 mutant, S71E-Pin1, using solution NMR. We find that S71E perturbs not only its host loop but also the nearby PPIase core. The perturbations identify a local network of hydrogen bonds and salt bridges that is more extended than previously thought, and includes interactions between the catalytic loop and the α2/α3 turn in the PPIase core. Explicit-solvent molecular dynamics simulations and phylogenetic analysis suggest that these interactions act as conserved "latches" between the loop and PPIase core that enhance binding of phosphorylated substrates, as they are absent in PPIases lacking pS/T-P specificity. Our results suggest that S71 is a hub residue within an electrostatic network primed for phosphorylation, and may illustrate a common mechanism of phosphorylation-mediated allostery.

Negative Regulation of Peptidyl-Prolyl Isomerase Activity by Interdomain Contact in Human Pin1.

Pin1 is a modular peptidyl-prolyl isomerase specific for phosphorylated Ser/Thr-Pro (pS/T-P) motifs, typically within intrinsically disordered regions 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 nuclear magnetic resonance 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.

Modeling conformational ensembles of slow functional motions in Pin1-WW.

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.

Dynamics of ligand binding from 13C NMR relaxation dispersion at natural abundance.

We show that Carr-Purcell-Meiboom-Gill (CPMG) 13Calpha NMR relaxation dispersion measurements are a viable means for profiling mus-ms ligand dynamics involved in receptor binding. Critically, the dispersion is at natural 13C abundance; this matches typical pharmaceutical research settings in which ligand isotope-labeling is often impractical. The dispersion reveals ligand 13Calpha nuclei that experience mus-ms modulation of their chemical shifts due to binding. 13Calpha shifts are dominated by local torsion angles , psi, chi1; hence, these experiments identify flexible torsion angles that may assist complex formation. Since the experiments detect the ligand, they are viable even in the absence of a receptor structure. The mus-ms dynamic information gained helps establish flexibility-activity relationships. We apply these experiments to study the binding of a phospho-peptide substrate ligand to the peptidyl-prolyl isomerase Pin1.

Direct 13C-detection for carbonyl relaxation studies of protein dynamics.

We describe a method that uses direct 13C-detection for measuring rotating-frame carbonyl (13CO) relaxation rates to describe protein functional dynamics. Key advantages of method include the following: (i) unique access to 13CO groups that lack a scalar-coupled 15N-1H group; (ii) insensitivity to 15N/1H exchange-broadening that can derail 1H-detected 15N and HNCO methods; (iii) avoidance of artifacts caused by incomplete water suppression. We demonstrate the approach for both backbone and side-chain 13CO groups. Accuracy of the 13C-detected results is supported by their agreement with those obtained from established HNCO-based approaches. Critically, we show that the 13C-detection approach provides access to the 13CO groups of functionally important residues that are invisible via 1H-detected HNCO methods because of exchange-broadening. Hence, the 13C-based method fills gaps inherent in canonical 1H-detected relaxation experiments, and thus provides a novel complementary tool for NMR studies of biomolecular flexibility.

Sequence-specific dynamics modulate recognition specificity in WW domains.

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.

Substrate recognition reduces side-chain flexibility for conserved hydrophobic residues in human Pin1.

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.