On the benefit of bivalency in peptide ligand/pin1 interactions

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO₃H₂)/Thr(PO₃H₂)-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.     

Coupled intra- and interdomain dynamics support domain cross-talk in Pin1

The functional mechanisms of multidomain proteins often exploit interdomain interactions, or “cross-talk.” An example is human Pin1, an essential mitotic regulator consisting of a Trp–Trp (WW) domain flexibly tethered to a peptidyl-prolyl isomerase (PPIase) domain, resulting in interdomain interactions important for Pin1 function. Substrate binding to the WW domain alters its transient contacts with the PPIase domain via means that are only partially understood. Accordingly, we have investigated Pin1 interdomain interactions using NMR paramagnetic relaxation enhancement (PRE) and molecular dynamics (MD) simulations. The PREs show that apo-Pin1 samples interdomain contacts beyond the range suggested by previous structural studies. They further show that substrate binding to the WW domain simultaneously alters interdomain separation and the internal conformation of the WW domain. A 4.5-μs all-atom MD simulation of apo-Pin1 suggests that the fluctuations of interdomain distances are correlated with fluctuations of WW domain interresidue contacts involved in substrate binding. Thus, the interdomain/WW domain conformations sampled by apo-Pin1 may already include a range of conformations appropriate for binding Pin1''s numerous substrates. The proposed coupling between intra-/interdomain conformational fluctuations is a consequence of the dynamic modular architecture of Pin1. Such modular architecture is common among cell-cycle proteins; thus, the WW–PPIase domain cross-talk mechanisms of Pin1 may be relevant for their mechanisms as well.     

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.     

Peptide binding induces large scale changes in inter-domain mobility in human Pin1

Pin1 is a peptidyl-prolyl cis/trans isomerase (PPIase) essential for cell cycle regulation. Pin1-catalyzed peptidyl-prolyl isomerization provides a key conformational switch to activate phosphorylation sites with the common phospho-Ser/Thr-Pro sequence motif. This motif is ubiquitously exploited in cellular response to a variety of signals. Pin1 is able to bind phospho-Ser/Thr-Pro-containing sequences at two different sites that compete for the same substrate. One binding site is located within the N-terminal WW domain, which is essential for protein targeting and localization. The other binding site is located in the C-terminal catalytic domain, which is structural homologous to the FK506-binding protein (FKBP) class of PPIases. A flexible linker of 12 residues connects the WW and catalytic domain. To characterize the structure and dynamics of full-length Pin1 in solution, high resolution NMR methods have been used to map the nature of interactions between the two domains of Pin1. In addition, the influence of target peptides on domain interactions has been investigated. The studies reveal a dynamic picture of the domain interactions. 15N spin relaxation data, differential chemical shift mapping, and residual dipolar coupling data indicate that Pin1 can either behave as two independent domains connected by the flexible linker or as a single intact domain with some amount of hinge bending motion depending on the sequence of the bound peptide. The functional importance of the modulation of relative domain flexibility in light of the multitude of interaction partners of Pin1 is discussed.     

An essential role for Pin1 in Xenopus laevis embryonic development revealed by specific inhibitors

The peptidyl prolyl cis/trans isomerase (PPIase) Pin1 plays an important role in phosphorylation-dependent events of the cell cycle. This function is linked to its display of two phosphothreonine/phosphoserine-proline binding motifs, one within the type IV WW domain and a second within the parvulin-like catalytic domain. By microinjection of the compound Ac-Phe-D-Thr(PO3H2)-Pip-Nal-Gln-NH2, which inhibits Xenopus laevis Pin1 with a Ki value of 19.4+/-1.5 nM, into the animal pole of X. laevis embryos at the two-cell stage, the impact of Pin1 PPIase activity on cell cycle progression and embryonic development could be analysed, independent of WW domain-mediated phosphoprotein binding. Injected embryos showed a dramatically decreased survival rate at late stages of development that could only be partially compensated by co-injection with mRNAs of enzymatically active Pin1 variants, demonstrating that the phosphorylation-specific PPIase activity of Pin1 is essential for cell division and development in X. laevis.     

Thermodynamics of phosphopeptide binding to the human peptidyl prolyl cis/trans isomerase Pin1

Proteins containing phosphorylated Ser/Thr-Pro motifs play key roles in numerous regulatory processes in the cell. The peptidyl prolyl cis/trans isomerase Pin1 specifically catalyzes the conformational transition of phosphorylated Ser/Thr-Pro motifs. Here we report the direct analysis of the thermodynamic properties of the interaction of the PPIase Pin1 with its substrate-analogue inhibitor Ac-Phe-D-Thr(PO3H2)-Pip-Nal-Gln-NH2 specifically targeted to the PPIase active site based on the combination of isothermal titration calorimetry and studies on inhibition of enzymatic activity of wt Pin1 and active site variants. Determination of the thermodynamic parameters revealed an enthalpically and entropically favored interaction characterized by binding enthalpy deltaH(ITC) of -6.3 +/- 0.1 kcal mol(-1) and a TdeltaS(ITC) of 4.1 +/- 0.1 kcal mol(-1). The resulting dissociation constant KD for binding of the peptidic inhibitor with 1.8 x 10(-8) M resembles the dissociation constant of a Pin1 substrate in the transition state, suggesting a transition state analogue conformation of the bound inhibitor. The strongly decreased affinity of Pin1 for ligand at increasing ionic strength implicates that the potential of bidentate binding of a substrate protein by the PPIase and the WW domain of Pin1 may be required to deploy improved efficiency and specificity of Pin1 under conditions of physiological ionic strength.     

Structural basis for phosphoserine-proline recognition by group IV WW domains

Pin1 contains an N-terminal WW domain and a C-terminal peptidyl-prolyl cis-trans isomerase (PPIase) domain connected by a flexible linker. To address the energetic and structural basis for WW domain recognition of phosphoserine (P.Ser)/phosphothreonine (P. Thr)- proline containing proteins, we report the energetic and structural analysis of a Pin1-phosphopeptide complex. The X-ray crystal structure of Pin1 bound to a doubly phosphorylated peptide (Tyr-P.Ser-Pro-Thr-P.Ser-Pro-Ser) representing a heptad repeat of the RNA polymerase II large subunit's C-terminal domain (CTD), reveals the residues involved in the recognition of a single P.Ser side chain, the rings of two prolines, and the backbone of the CTD peptide. The side chains of neighboring Arg and Ser residues along with a backbone amide contribute to recognition of P.Ser. The lack of widespread conservation of the Arg and Ser residues responsible for P.Ser recognition in the WW domain family suggests that only a subset of WW domains can bind P.Ser-Pro in a similar fashion to that of Pin1.     

Cyclohexyl Ketone Inhibitors of Pin1 Dock in a Trans-Diaxial Cyclohexane Conformation

Cyclohexyl ketone substrate analogue inhibitors (Ac–pSer-Ψ[C = OCH]-Pip–tryptamine) of Pin1, the cell cycle regulatory peptidyl-prolyl isomerase (PPIase), were designed and synthesized as potential electrophilic acceptors for the Pin1 active site Cys113 nucleophile to test a proposed nucleophilic addition-isomerization mechanism. Because they were weak inhibitors, models of all three stereoisomers were docked into the active site of Pin1. Each isomer consistently minimized to a trans-diaxial cyclohexane conformation. From this, we hypothesize that Pin1 stretches substrates into a trans-pyrrolidine conformation to lower the barrier to isomerization. Our reduced amide inhibitor of Pin1 adopted a similar trans-pyrrolidine conformation in the crystal structure. The molecular model of 1, which mimics the l-Ser-l-Pro stereochemistry, in the Pin1 active site showed a distance of 4.4 Å, and an angle of 31° between Cys113-S and the ketone carbon. The computational models suggest that the mechanism of Pin1 PPIase is not likely to proceed through nucleophilic addition.     

The dual histidine motif in the active site of Pin1 has a structural rather than catalytic role

The catalytic domain of the peptidyl-prolyl cis/ trans isomerase Pin1 is a member of the FKBP superfold family. Within its active site are two highly conserved histidine residues, H59 and H157. Despite their sequence conservation in parvulin PPIase domains, the role of these histidine residues remains unclear. Our previous work (Behrsin et al. (2007) J. Mol. Biol. 365, 1143- 1162.) was consistent with a model where one or both histidines had critical roles in a hydrogen bonding network in the active site. Here, we test this model by looking at the effect of mutations to H59 and H157 on Pin1 function, activity, and protein stability. Using a yeast complementation assay, we show that both H59 and H157 can be mutated to non-hydrogen bonding residues and still support viability. Surprisingly, a nonfunctional H59L mutation can be rescued by a mutation of H157, to leucine. This double mutation (H59L/H157L) also had about 5-fold greater isomerase activity than the H59L mutation with a phosphorylated substrate. Structural analyses suggest that rescue of function and activity results from partial rescue of protein stability. Our findings indicate that H59 and H157 are not required for hydrogen bonding within the active site, and in contrast to the active site C113, they do not participate directly in catalysis. Instead, we suggest these histidines play a key role in domain structure or stability.     

The structure of apo protein-tyrosine phosphatase 1B C215S mutant: more than just an S --> O change

Protein-tyrosine phosphatases catalyze the hydrolysis of phosphate monoesters via a two-step mechanism involving a covalent phospho-enzyme intermediate. Biochemical and site-directed mutagenesis experiments show that the invariant Cys residue present in the PTPase signature motif (H/V)CX(5)R(S/T) (i.e., C215 in PTP1B) is absolutely required for activity. Mutation of the invariant Cys to Ser results in a catalytically inactive enzyme, which still is capable of binding substrates and inhibitors. Although it often is assumed that substrate-trapping mutants such as the C215S retain, in solution, the structural and binding properties of wild-type PTPases, significant differences have been found in the few studies that have addressed this issue, suggesting that the mutation may lead to structural/conformational alterations in or near the PTP1B binding site. Several crystal structures of apo-WT PTP1B, and of WT- and C215S-mutant PTP1B in complex with different ligands are available, but no structure of the apo-PTP1B C215S has ever been reported. In all previously reported structures, residues of the PTPase signature motif have an identical conformation, while residues of the WPD loop (a surface loop which includes the catalytic Asp) assume a different conformation in the presence or absence of ligand. These observations led to the hypothesis that the different spectroscopic and thermodynamic properties of the mutant protein may be the result of a different conformation for the WPD loop. We report here the structure of the apo-PTP1B C215S mutant, which reveals that, while the WPD loop is in the open conformation observed in the apo WT enzyme crystal structure, the residues of the PTPases signature motif are in a dramatically different conformation. These results provide a structural basis for the differences in spectroscopic properties and thermodynamic parameters in inhibitor binding observed for the wild-type and mutant enzymes.     

Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.     

Role of Asp187 and Gln190 in the Na+/proline symporter (PutP) of Escherichia coli

Asp187 and Gln190 were predicted as conserved and closely located at the Na(+) binding site in a topology and homology model structure of Na(+)/proline symporter (PutP) of Escherichia coli. The replacement of Asp187 with Ala or Leu did not affect proline transport activity; whereas, change to Gln abolished the active transport. The binding affinity for Na(+) or proline of these mutants was similar to that of wild-type (WT) PutP. This result indicates Asp187 to be responsible for active transport of proline without affecting the binding. Replacement of Gln190 with Ala, Asn, Asp, Leu and Glu had no effect on transport or binding, suggesting that it may not have a role in the transport. However, in the negative D187Q mutant, a second mutation, of Gln190 to Glu or Leu, restored 46 or 7% of the transport activity of WT, respectively, while mutation to Ala, Asn or Asp had no effect. Thus, side chain at position 190 has a crucial role in suppressing the functional defect of the D187Q mutant. We conclude that Asp187 is responsible for transport activity instead of coupling-ion binding by constituting the translocation pathway of the ion and Gln190 provides a suppressing mutation site to regain PutP functional activity.     

Residue 219 impacts on the dynamics of the C-terminal region in glutathione transferase A1-1: implications for stability and catalytic and ligandin functions

The C-terminal region in class alpha glutathione transferases (GSTs) modulates the catalytic and nonsubstrate ligand binding functions of these enzymes. Except for mouse GST A1-1 (mGST A1-1), the structures of class alpha GSTs have a bulky aliphatic side chain topologically equivalent to Ile219 in human GST A1-1 (hGST A1-1). In mGST A1-1, the corresponding residue is an alanine. To investigate the role of Ile219 in determining the conformational dynamics of the C-terminal region in hGST A1-1, the residue was replaced by alanine. The substitution had no effect on the global structure of hGST A1-1 but did reduce the conformational stability of the C-terminal region of the protein. This region could be stabilized by ligands bound at the active site. The catalytic behavior of hGST A1-1 was significantly compromised by the I219A mutation as demonstrated by reduced enzyme activity, increased K(m) for the substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), and reduced catalytic efficiencies. Inhibition studies also indicated that the binding affinities for product and substrate analogues were dramatically decreased. The affinity of the mutant for GSH was, however, only slightly increased, indicating that the G-site was unaltered by the mutation. The binding affinity and stoichiometry for the anionic dye 8-anilino-1-naphthalene sulfonate (ANS) was also not significantly affected by the I219A mutation. However, the lower DeltaC(p) for ANS binding to the mutant (-0.34 kJ/mol per K compared with -0.84 kJ/mol per K for the wild-type protein) suggests that ANS binding to the mutant results in the burial of less hydrophobic surface area. Fluorescence data also indicates that ANS bound to the mutant is more prone to quenching by water. Overall, the data from this study, together with the structural details of the C-terminal region in mGST A1-1, show that Ile219 is an important structural determinant of the stability and dynamics of the C-terminal region of hGST A1-1.     

An irreversible inhibitor of peptidyl-prolyl cis/trans isomerase Pin1 and evaluation of cytotoxicity

Pin1 (protein interacting with never in mitosis A-1) is a member of the peptidyl prolyl isomerase (PPIase) family, and catalyzes cis-trans isomerization of pThr/Ser-Pro amide bonds. Because Pin1 is overexpressed in various cancer cell lines and promotes cell growth, it is considered a target for anticancer agents. Here, we designed and synthesized a covalently binding Pin1 inhibitor (S)-2 to target Pin1’s active site. This compound inhibited Pin1 in protease-coupled assay, and formed a covalent bond with Cys113 of Pin1, as determined by ESI-MS. The acetoxymethyl ester of (S)-2, i.e., 6, suppressed cyclin D1 expression in human prostate cancer PC-3 cells, and exhibited cytotoxicity. Pin1-knockdown experiments indicated that a target for the cytotoxicity of 6 is Pin1.     

Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function

The microtubule binding protein gephyrin plays a prominent role in establishing and maintaining a high concentration of inhibitory glycine receptors juxtaposed to presynaptic releasing sites. Here, we show that endogenous gephyrin undergoes proline-directed phosphorylation, which is followed by the recruitment of the peptidyl-prolyl isomerase Pin1. The interaction between gephyrin and Pin1 is strictly dependent on gephyrin phosphorylation and requires serine-proline consensus sites encompassing the gephyrin proline-rich domain. Upon binding, Pin1 triggers conformational changes in the gephyrin molecule, thus enhancing its ability to bind the beta subunit of GlyRs. Consistently, a downregulation of GlyR clusters was detected in hippocampal neurons derived from Pin1 knockout mice, which was paralleled by a reduction in the amplitude of glycine-evoked currents. Our results suggest that phosphorylation-dependent prolyl isomerisation of gephyrin represents a mechanism for regulating GlyRs function.     

Three-dimensional structure of horse liver alcohol dehydrogenase at 2-4 A resolution.

The crystal structure analysis of horse liver alcohol dehydrogenase has been extended to 2.4 Å resolution. From the corresponding electron density map of the apoenzyme we have determined the positions of the 374 amino acids in the polypeptide chain of each subunit.The coenzyme binding domain of the subunit comprises residues 176 to 318. 45% of these residues are helical and 32% are in the central six-stranded pleated sheet structure. The positions and orientations of the helices with respect to the pleated sheet indicate a possible folding mechanism for this part of the subunit structure. The coenzyme analogue ADP-ribose binds to this domain in a position and orientation very similar to coenzyme binding to lactate dehydrogenase. The adenine part binds in a hydrophobic pocket, the adenosine ribose is hydrogen-bonded to the side chain of Asp223, the pyrophosphate is positioned by interaction with Arg47 and the nicotinamide ribose is 6Å away from the catalytic zinc atom.The catalytic domain is mainly built up from three distinct antiparallel pleated-sheet regions. Residues within this domain provide ligands to the catalytic zinc atom; Cys46, His67 and Cys174. An approximate tetrahedral coordination of this zinc is completed by a water molecule or hydroxyl ion depending on the pH. Residues 95 to 113 form a lobe that binds the second zinc atom of the subunit. This zinc is liganded in a distorted tetrahedral arrangement by four sulphur atoms from the cysteine residues 97, 100, 103 and 111. The lobe forms one side of a significant cleft in the enzyme surface suggesting that this region might constitute a second catalytic centre of unknown function.The two domains of the subunit are separated by a crevice that contains a wide and deep hydrophobic pocket. The catalytic zinc atom is at the bottom of this pocket, with the zinc-bound water molecule projecting out into the pocket. This water molecule is hydrogen-bonded to the side chain of Ser48 which in turn is hydrogen-bonded to His51. The pocket which in all probability is the binding site for the substrate and the nicotinamide moiety of the coenzyme, is lined almost exclusively with hydrophobic side chains. Both subunits contribute residues to each of the two substrate binding pockets of the molecule. The only accessible polar groups in the vicinity of the catalytic centre are Ser48 and Thr178 apart from zinc and the zinc-bound water molecule.     

Solution structure of the His12 --> Cys mutant of the N-terminal zinc binding domain of HIV-1 integrase complexed to cadmium.

The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex.     

A mutation in the lactose permease of Escherichia coli that decreases conformational flexibility and increases protein stability

Lactose permease with Cys154 --> Gly (helix V) binds substrate with high affinity but catalyzes little or no transport. The purified, detergent-solubilized mutant protein exhibits much greater thermal stability than the wild type and little tendency to aggregate. Stabilization is also observed in vivo with an unstable mutant that is expressed at significantly higher levels when the Cys154 --> Gly mutation is introduced. In addition, ligand-induced conformational changes are markedly reduced or abolished by the Cys154 --> Gly mutation: (i) Although the fluorescence of purified single Trp33 (helix I) permease is enhanced by ligand binding, introduction of the Cys154 --> Gly mutation abolishes the effect. (ii) The rate of 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) labeling of permease with a single Cys residue in place of Val331 (helix X) is increased in the presence of ligand but reduced when the Cys154 --> Gly mutation is present. (iii) Fluorescence emission intensity of MIANS-labeled single Cys331 permease is enhanced and blue shifted in the Cys154 --> Gly mutant background, indicating that the latter mutation causes position 331 to become exposed to a less polar environment. The results indicate that the Cys154 --> Gly mutation causes a more compact structure and decreased conformational flexibility, an alteration that specifically blocks the structural changes necessary for substrate translocation with little or no effect on ligand binding.     

Allele-specific activation of genetically engineered receptors.

The binding of agonists and antagonists to the beta-adrenergic receptor (beta AR) is postulated to involve an ionic interaction between the amine group of the ligand and the carboxylate side chain of Asp113 in the third hydrophobic domain of the receptor. To explore the importance of this interaction in the binding of ligands to the beta AR, a Ser residue was substituted for Asp113, and the ability of this mutant receptor to respond to compounds which could potentially interact with the hydroxyl side chain of the Ser residue was assessed. The mutant receptor was fully activated by catechol-containing esters and ketones, compounds which did not activate the wild-type beta AR. The demonstration that the molecular substitution of a single amino acid residue can alter the ligand binding specificity of the beta AR provides evidence that the chemical nature of this residue is a critical determinant in the recognition site of the receptor. Further, the ability to modify the specificity of a receptor by the replacement of amino acids at the binding site demonstrates the potential for the rational design of drugs which function specifically at genetically engineered receptors.