Pin1 is a modular enzyme that accelerates the cis-trans isomerization of phosphorylated-Ser/Thr-Pro (pS/T-P) motifs found in numerous signaling proteins regulating cell growth and neuronal survival. We have used NMR to investigate the interaction of Pin1 with three related ligands that include a pS-P substrate peptide, and two pS-P substrate analogue inhibitors locked in the cis and trans conformations. Specifically, we compared the ligand binding modes and binding-induced changes in Pin1 side-chain flexibility. The cis and trans binding modes differ, and produce different mobility in Pin1. The cis-locked inhibitor and substrate produced a loss of side-chain flexibility along an internal conduit of conserved hydrophobic residues, connecting the domain interface with the isomerase active site. The trans-locked inhibitor produces a weaker conduit response. Thus, the conduit response is stereoselective. We further show interactions between the peptidyl-prolyl isomerase and Trp-Trp (WW) domains amplify the conduit response, and alter binding properties at the remote peptidyl-prolyl isomerase active site. These results suggest that specific input conformations can gate dynamic changes that support intraprotein communication. Such gating may help control the propagation of chemical signals by Pin1, and other modular signaling proteins.allostery | protein dynamics | ligand dynamics | protein evolution P hospho-serine/threonine-proline (pS/T-P) motifs are signaling motifs within intrinsically disordered loops of cell cycle proteins (1). The imide bond between the pS/T and P residues can adopt either the cis or trans conformation. These conformations differ in their susceptibility to kinases and phosphatases that propagate the chemical signals governing the cell cycle. Accordingly, the cell must regulate the cis/trans populations of these pS/T-P motifs to ensure proper signal routing.In this context, the peptidyl-prolyl isomerase Pin1 has emerged as a critical regulator (2, 3). Pin1 is a reversible enzyme that catalyzes the cis-trans isomerization of the pS/T-P imide linkages (2, 3) of other signaling proteins, such as CDC25C, p53, c-Myc, NF-kB, cyclin D1, and tau (3). Pin1 engages when external events, such as S/T (de)-phosphorylation, change the cis-trans equilibrium. Pin1 then catalyzes the cis-trans isomerization, thereby accelerating the approach to the new equilibrium (1).Pin1 is a modular protein of 163 residues consisting of a WW domain (1-39) and a larger peptidyl-prolyl isomerase (PPIase) domain (50-163) (Fig. 1). A flexible linker connects the two domains. Both domains are specific for pS/T-P motifs (1). The WW domain serves as a docking module, whereas catalysis is the sole province of the PPIase domain. Earlier structural studies of Pin1 revealed conformational changes upon substrate interaction, thus motivating flexibility-function studies of Pin1 (4-6). Our previous NMR deuterium relaxation studies of Pin1 mapped the changes in flexibility of methyl-bearing side chains caused by interaction with an establi...

The mechanism of the cell cycle regulatory peptidyl prolyl isomerase (PPIase), Pin1, was investigated using reduced-amide inhibitors designed to mimic the twisted-amide transition state. Inhibitors, R–pSer–Ψ[CH2N]–Pro–2-(indol-3-yl)-ethylamine, 1 (R = fluorenylmethoxycarbonyl, Fmoc), and 2 (R = Ac), of Pin1 were synthesized and bioassayed. Inhibitor 1 had an IC50 value of 6.3 μM, which is 4.5-fold better inhibition for Pin1 than our comparable ground state analogue, a cis-amide alkene isostere containing inhibitor. The change of Fmoc to Ac in 2 improved aqueous solubility for structural determination, and resulted in an IC50 value of 12 μM. The X-ray structure of the complex of 2 bound to Pin1 was determined to 1.76 Å resolution. The structure revealed that the reduced amide adopted a conformation similar to the proposed twisted-amide transition state of Pin1, with a trans-pyrrolidine conformation of the prolyl ring. A similar conformation of substrate would be destabilized relative to the planar amide conformation. Three additional reduced amides, with Thr replacing Ser, and l- or d-pipecolate (Pip) replacing Pro, were slightly weaker inhibitors of Pin1.

Flexible ligands are common starting points in drug discovery. Such ligands often change their conformational flexibility upon receptor interaction. Mapping these changes can help guide the manipulation of ligand flexibility in molecular design. To gain this insight, we need methods that are sensitive to (1) ligand motions related to receptor activity such as binding or catalysis and (2) ligand modifications that alter the activity-related motions.Here we demonstrate an approach that satisfies these requirements: ligand flexibilityactivity studies by Nuclear Magnetic Resonance (NMR). These studies apply dynamic NMR experiments to a series of related ligands that target a common receptor and compare the site-specific changes in ligand dynamics stimulated by receptor interaction. The comparisons reveal how variations in ligand structure can perturb ligand motions important for activity and, thus, provide site-specific information for changing ligand mobility (e.g., rigidification). This systematic approach for articulating activity-related ligand dynamics is a critical step toward establishing flexibility-activity relationships (FAR), to complement standard structure-activity relationships (SAR) in iterative design.We illustrate these NMR FAR-related methods by studying ligands with motions that are (1) intrinsic to protein interaction and (2) sensitive to structural modifications. Specifically, we examine three structurally similar but dynamically differentiated ligands of human Pin1 (Scheme 1). Pin1 is a mitotic regulator that accelerates the cis-trans isomerization of phospho-Ser/Thr-Pro motifs of other signaling proteins. 1,2 The ligands include a phosphopeptide substrate, Ac-Phe-Phe-pSer-Pro-Arg-NH 2 (FF-pSPR), and two inhibitor analogues. The analogues replace the core peptidyl-prolyl linkage (pSP) by alkene isosteres, resulting in cis-locked (Ac-Phe-Phe-pSer-Ψ[(Z)CH=C]-Pro-Arg-NH 2 ) or trans-locked (AcPhe-Phe-pSer-Ψ[(E)CH=C]-Pro-Arg-NH 2 ) peptidic ligands that are competitive Pin1 inhibitors. 3,4 We exploit the sensitivity of aliphatic 13 C chemical shifts to local conformation. [5][6][7][8] Receptor interactions can dynamically alter ligand conformation, modulating the ligand 13 C shifts, thus causing 13 C relaxation dispersion. To map ligand dynamics site-specifically, we © 2010 American Chemical Society Correspondence to: Jeffrey W. Peng, jpeng@nd.edu. Supporting Information Available: Examples of 2-D 13 C-1 H dispersion spectra; exchange parameters for the trans-locked inhibitor; figure of binding isotherms; dispersion for all three isolated ligands at 295 K; dispersion at multiple temperatures and protein concentrations; 2-D EXSY of the FFpSPR substrate; details of sample preparation, dispersion measurements, and model fitting. This material is available free of charge via the Internet at http://pubs.acs.org. The dispersion experiments detect the modulation of the 13 C chemical shifts caused by exchange dynamics. The modulation enhances the 13 C transverse relaxation (line broadening), which becomes ...

Repeat proteins have recently emerged as especially well-suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence-structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand-recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N-and C-capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand.