Everolimus therapy was associated with marked reduction in the volume of subependymal giant-cell astrocytomas and seizure frequency and may be a potential alternative to neurosurgical resection in some cases, though long-term studies are needed. (Funded by Novartis; ClinicalTrials.gov number, NCT00411619.).

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...

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Pin1 is an essential mitotic regulator consisting of a peptidyl–prolyl isomerase (PPIase) domain flexibly tethered to a smaller Trp–Trp (WW) binding domain. Communication between these domains is important for Pin1 in vivo activity; however, the atomic basis for this communication has remained elusive. Our previous nuclear magnetic resonance (NMR) studies of Pin1 functional dynamics suggested that weak interdomain contacts within Pin1 enable allosteric communication between the domain interface and the distal active site of the PPIase domain.1,2 A necessary condition for this hypothesis is that the intrinsic properties of the PPIase domain should be sensitive to interdomain contact. Here, we test this sensitivity by generating a Pin1 mutant, I28A, which weakens the wild-type interdomain contact while maintaining the overall folds of the two domains. Using NMR, we show that I28A leads to altered substrate binding affinity and isomerase activity. Moreover, I28A causes long-range perturbations to conformational flexibility in both domains, for both the apo and substrate-complexed states of the protein. These results show that the distribution of conformations sampled by the PPIase domain is sensitive to interdomain contact and strengthen the hypothesis that such contact supports interdomain allosteric communication in Pin1. Other modular systems may exploit interdomain interactions in a similar manner.

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