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1.
Curr Protoc ; 4(3): e1010, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38516989

ABSTRACT

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami et al. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNASerPro.


Subject(s)
Proline , Serine , Proline/chemistry , RNA, Transfer/chemistry , RNA, Transfer/genetics , RNA, Transfer/metabolism , Amino Acids/chemistry , Amino Acids/metabolism , Dipeptides , Peptides
2.
Org Biomol Chem ; 21(19): 4039-4051, 2023 05 17.
Article in English | MEDLINE | ID: mdl-37114339

ABSTRACT

We report the first experimental evidence for a fluoro-alkene amide isostere participating in n→π* donation, which stabilizes the collagen triple helix. Of the three amide positions in canonical collagen-like peptides, Gly-Pro, Pro-Hyp, and Hyp-Gly, triple helix stability stands to benefit from substitution of only the isomerizable 3° Gly-Pro amide bond with a trans-locked fluoro-alkene. A (Z)-fluoro-alkene isostere of Gly-trans-Pro was synthesized, and its effect on the thermostability of a collagen-like peptide triple helix was measured. The mixture of enantiomers, Boc-Gly-Ψ[(Z)CFC]-L/D-Pro-OH, was synthesized in 8 steps with 27% overall yield, and the Fmoc-Gly-Ψ[(Z)CFC]-L/D-Pro-Hyp-OBn diastereomers were separated. The Gly-Ψ[(Z)CFC]-Pro isostere installed in a collagen-like peptide forms a stable triple helix. By CD, the thermal melting (Tm) value of the fluoro-alkene peptide was +42.2 ± 0.4 °C, and the Tm value of the control peptide was +48.4 ± 0.5 °C, a difference in stability of ΔTm -6.2 °C. Deshielding of the fluorine nucleus in the 19F NMR spectra is evidence of a stabilizing n→π* electronic interaction.


Subject(s)
Alkenes , Peptides , Peptides/chemistry , Amides , Collagen/chemistry , Protein Conformation
3.
Angew Chem Int Ed Engl ; 62(2): e202209768, 2023 01 09.
Article in English | MEDLINE | ID: mdl-36480758

ABSTRACT

Full integration of green chemistry into the undergraduate curriculum is a necessity to prepare our students for a sustainable future. We discuss the reasons for the need to change the curriculum, the institutions in North America, Europe, and Asia that are leading the way towards integration with classroom resources, and the published textbooks that are currently available for both classroom and laboratory. We plead for more time for hard-pressed college professors to revamp the curriculum, and for these efforts to be valued. We feel compelled by the urgency of this need to implore the chemistry education community to participate in these efforts now.


Subject(s)
Curriculum , Humans
4.
J Phys Chem B ; 126(1): 217-228, 2022 01 13.
Article in English | MEDLINE | ID: mdl-34968406

ABSTRACT

Collagen is the most abundant human protein, with the canonical sequence (Gly-Pro-Hyp)n in its triple helix region. Cis-trans isomerization of the Xaa-Pro amide has made two of these amide bonds the target of alkene replacement: the Gly-Pro and the Pro-Hyp positions. The conformations of Gly-Pro and Pro-Pro (as a Pro-Hyp model) fluoro-, chloro-, and proteo-alkene mimic models were investigated computationally to determine whether these alkenes can stabilize the polyproline type II (PPII) conformation of collagen. Second-order Møller-Plesset (MP2) calculations with various basis sets were used to perform the conformational analyses and locate stationary points. The calculation results predict that fluoro- and chloro-alkene mimics of Gly-Pro and Pro-Pro can participate in n→π* donation to stabilize PPII conformations, yet they are poor n→π* acceptors, shifting the global minima away from PPII conformations. For the proteo-alkene mimics, the lack of significant n→π* interactions and unstable PPII-like geometries explains their known destabilization of the triple helix in collagen-like peptides.


Subject(s)
Alkenes , Collagen , Dipeptides , Humans , Protein Conformation , Protein Structure, Secondary
5.
Protein Sci ; 30(4): 804-817, 2021 04.
Article in English | MEDLINE | ID: mdl-33512005

ABSTRACT

Designed protein receptors hold diagnostic and therapeutic promise. We now report the design of five consensus leucine-rich repeat proteins (CLRR4-8) based on the LRR domain of nucleotide-binding oligomerization domain (NOD)-like receptors involved in the innate immune system. The CLRRs bind muramyl dipeptide (MDP), a bacterial cell wall component, with micromolar affinity. The overall Kd app values ranged from 1.0 to 57 µM as measured by fluorescence quenching experiments. Biphasic fluorescence quenching curves were observed in all CLRRs, with higher affinity Kd1 values ranging from 0.04 to 4.5 µM, and lower affinity Kd2 values ranging from 3.1 to 227 µM. These biphasic binding curves, along with the docking studies of MDP binding to CLRR4, suggest that at least two MDPs bind to each protein. Previously, only single MDP binding was reported. This high-capacity binding of MDP promises small, soluble, stable CLRR scaffolds as candidates for the future design of pathogen biosensors.


Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/chemistry , Proteins/chemistry , Humans , Leucine-Rich Repeat Proteins , Protein Binding , Proteins/chemical synthesis
6.
J Phys Chem B ; 123(2): 496-503, 2019 01 17.
Article in English | MEDLINE | ID: mdl-30525631

ABSTRACT

Ab initio calculations of three models of collagen at positions Pro-Pro-Gly (1), Pro-Gly-Pro (2), and Gly-Pro-Pro (3) were performed to assess the conformational variation of n→π* contributions to the stability of the collagen triple helix. Full conformational analyses by relaxed potential-energy scans of the Ψ dihedral angle of the central residue in models 1, 2, and 3 revealed the presence of several n→π* interactions. In model 2, with Gly as the central residue, both the Φ and Ψ dihedral angles of Gly were scanned. Most minima of each model contained one or two n→π* interactions, with pyramidalization at the π* carbon. We also observed pyramidalization at the n→π* donor amide nitrogens. Minima with hydrogen-bond or non-native n→π* interactions compete with the collagen stabilizing n→π* interactions. The collagen-like n→ re-π* conformation was found as the global minimum only in model 3. The global minimum of 1 had a 5-membered ring hydrogen bond with an additional weak n→ si-π* interaction. The global minimum of 2 was in the extended conformation. We predict that the n→π* interactions found in native collagen, while individually small, cumulatively contribute to the stability of the triple helix conformation of collagen.


Subject(s)
Collagen/chemistry , Hydrogen Bonding , Models, Chemical , Models, Molecular , Protein Conformation, alpha-Helical , Protein Structure, Quaternary , Static Electricity , Thermodynamics
7.
PLoS One ; 10(10): e0139543, 2015.
Article in English | MEDLINE | ID: mdl-26445009

ABSTRACT

Three stereoisomeric inhibitors of Pin1: (2R,5S)-, (2S,5R)- and (2S,5S)-Ac-pSer-Ψ[(Z)CH = C]-pipecolyl(Pip)-2-(2-naphthyl)ethylamine 1, that mimic L-pSer-D-Pro, D-pSer-L-Pro, and D-pSer-D-Pro amides respectively, were synthesized by a 13-step route. The newly formed stereogenic centers in the pipecolyl ring were introduced by Luche reduction, followed by stereospecific [2,3]-Still-Wittig rearrangement. The (Z)- to (E)-alkene ratio in the rearrangements were consistently 5.5 to 1. The stereochemistry at the original Ser α-carbon controlled the stereochemistry of the Luche reduction, but it did not affect the stereochemical outcome of the rearrangement, which consistently gave the (Z)-alkene. The epimerized by-product, (2S,5S)-10, resulting from the work-up after Na/NH3 debenzylation of (2S,5R)-9, was carried on to the (2S,5S)-1 isomer. Compound (2S,5S)-10 was resynthesized from the Luche reduction by-product, (2R,3R)-3, and the stereochemistry was confirmed by comparison of the optical rotations. The IC50 values for (2R,5S)-1, (2S,5R)-1 and (2S,5S)-1 Pin1 inhibition were: 52, 85, and 140 µM, respectively.


Subject(s)
Alkenes/chemistry , Amides/chemistry , Peptidylprolyl Isomerase/antagonists & inhibitors , Biochemical Phenomena/physiology , Molecular Structure , Stereoisomerism
8.
ACS Chem Biol ; 10(10): 2405-14, 2015 Oct 16.
Article in English | MEDLINE | ID: mdl-26332362

ABSTRACT

Proline isomerization greatly impacts biological signaling but is subtle and difficult to detect in proteins. We characterize this poorly understood regulatory mechanism for RNA polymerase II carboxyl terminal domain (CTD) phosphorylation state using novel, direct, and quantitative chemical tools. We determine the proline isomeric preference of three CTD phosphatases: Ssu72 as cis-proline specific, Scp1 and Fcp1 as strongly trans-preferred. Due to this inherent characteristic, these phosphatases respond differently to enzymes that catalyze the isomerization of proline, like Ess1/Pin1. We demonstrate that this selective regulation of RNA polymerase II phosphorylation state exists within human cells, consistent with in vitro assays. These results support a model in which, instead of a global enhancement of downstream enzymatic activities, proline isomerases selectively boost the activity of a subset of CTD regulatory factors specific for cis-proline. This leads to diversified phosphorylation states of CTD in vitro and in cells. We provide the chemical tools to investigate proline isomerization and its ability to selectively enhance signaling in transcription and other biological contexts.


Subject(s)
Models, Biological , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/metabolism , Proline/chemistry , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Animals , Blotting, Western , Crystallography, X-Ray , Drosophila/enzymology , Enzyme Activation/physiology , Humans , Isomerism , Molecular Structure
9.
ACS Chem Biol ; 10(4): 952-6, 2015 Apr 17.
Article in English | MEDLINE | ID: mdl-25603287

ABSTRACT

The cis vs trans conformation, or shape, of phosphoserine-proline (pSer-Pro), a prevalent motif in cell cycle proteins, may play a significant role in regulating mitosis. We demonstrate that Cdk1-cyclin B, the central mitotic kinase, is specific for the trans conformation, not cis, of synthetic, locked Ser-Pro 11-residue peptide substrates, using LC-MSMS detection and sequencing of phosphorylated products. This substrate stereospecificity may contribute an additional level of mitotic regulation.


Subject(s)
Cyclin B1/chemistry , Cyclin B1/metabolism , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/metabolism , CDC2 Protein Kinase , Mitosis , NIMA-Interacting Peptidylprolyl Isomerase , Peptides/chemistry , Peptides/metabolism , Peptidylprolyl Isomerase/metabolism , Phosphorylation , Protein Conformation , Solid-Phase Synthesis Techniques , Stereoisomerism , Tandem Mass Spectrometry
10.
Environ Monit Assess ; 186(6): 3717-24, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24497082

ABSTRACT

Disinfection by-products (DBPs) arise when natural organic matter in source water reacts with disinfectants used in the water treatment process. Studies have suggested an association between DBPs and birth defects. Neural tube defects (NTDs) in embryos of untreated control mice were first observed in-house in May 2006 and have continued to date. The source of the NTD-inducing agent was previously determined to be a component of drinking water. Tap water samples from a variety of sources were analyzed for trihalomethanes (THMs) to determine if they were causing the malformations. NTDs were observed in CD-1 mice provided with treated and untreated surface water. Occurrence of NTDs varied by water source and treatment regimens. THMs were detected in tap water derived from surface water but not detected in tap water derived from a groundwater source. THMs were absent in untreated river water and laboratory purified waters, yet the percentage of NTDs in untreated river water were similar to the treated water counterpart. These findings indicate that THMs were not the primary cause of NTDs in the mice since the occurrence of NTDs was unrelated to drinking water disinfection.


Subject(s)
Drinking Water/chemistry , Neural Tube Defects/chemically induced , Water Purification/methods , Animals , Disinfectants/toxicity , Disinfection/methods , Environmental Monitoring , Groundwater/chemistry , Male , Mice , Trihalomethanes/toxicity , Water Pollutants, Chemical/toxicity
11.
Biochemistry ; 52(44): 7707-13, 2013 Nov 05.
Article in English | MEDLINE | ID: mdl-24116866

ABSTRACT

The Pin1 peptidyl-prolyl isomerase catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac-Phe-Phe-phosphoSer-Pro-Arg-p-nitroaniline, were synthesized in unlabeled form, and with deuterium-labeled Ser-d3 and Pro-d7 amino acids. Kinetic data were collected as a function of Pin1 concentration to measure kinetic isotope effects (KIEs) on catalytic efficiency (kcat/Km). The normal secondary (2°) KIE value measured for the Ser-d3 substrate (kH/kD = 1.6 ± 0.2) indicates that the serine carbonyl does not rehybridize from sp(2) to sp(3) in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2° KIE can be explained by hyperconjugation between Ser α-C-H/D and C═O and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2° KIE value (kH/kD = 0.86 ± 0.08) measured for the Pro-d7 substrate indicates rehybridization of the prolyl nitrogen from sp(2) to sp(3) during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (kH2O/kD2O = 0.92 ± 0.12), indicating little or no involvement of exchangeable protons in the mechanism. These results support the formation of a simple twisted amide transition state as the mechanism for peptidyl prolyl isomerization catalyzed by Pin1. A model of the reaction mechanism is presented using crystal structures of Pin1 with ground state analogues and an inhibitor that resembles a twisted amide transition state.


Subject(s)
Amides/chemistry , Cytoplasmic Dyneins/chemistry , Deuterium/chemistry , Isomerism , Isotope Labeling , Kinetics , Peptides/chemistry , Substrate Specificity
12.
Cell ; 154(3): 637-50, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23911326

ABSTRACT

Synaptic plasticity induced by cocaine and other drugs underlies addiction. Here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. In response to D1-dopamine-receptor signaling that is induced by drug administration, the glutamate-receptor protein metabotropic glutamate receptor 5 (mGluR5) is phosphorylated by microtubule-associated protein kinase (MAPK), which we show potentiates Pin1-mediated prolyl-isomerization of mGluR5 in instances where the product of an activity-dependent gene, Homer1a, is present to enable Pin1-mGluR5 interaction. These biochemical events potentiate N-methyl-D-aspartate receptor (NMDAR)-mediated currents that underlie synaptic plasticity and cocaine-evoked motor sensitization as tested in mice with relevant mutations. The findings elucidate how a coincidence of signals from the nucleus and the synapse can render mGluR5 accessible to activation with consequences for drug-induced dopamine responses and point to depotentiation at corticostriatal synapses as a possible therapeutic target for treating addiction.


Subject(s)
Cocaine-Related Disorders/physiopathology , Cocaine/metabolism , Dopamine/metabolism , Peptidylprolyl Isomerase/metabolism , Amino Acid Sequence , Animals , Brain/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Embryo, Mammalian/metabolism , Homer Scaffolding Proteins , Long-Term Potentiation , Mice , Molecular Sequence Data , NIMA-Interacting Peptidylprolyl Isomerase , Phosphorylation , Receptors, AMPA/metabolism , Receptors, Dopamine D1/metabolism , Receptors, Kainic Acid/chemistry , Receptors, Kainic Acid/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Synapses/metabolism
13.
Peptides ; 41: 101-6, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23036324

ABSTRACT

The CAP2b neuropeptide family plays an important role in the regulation of the processes of diuresis and/or antidiuresis in a variety of insects. While Manse-CAP2b (pELYAFPRV-NH2) and native CAP2bs elicit diuretic activity in a number of species of flies, native CAP2b sequences have been shown to elicit antidiuretic activity in the kissing bug Rhodnius prolixus and the green stink bug Acrosternum hilare, the latter being an important pest of cotton and soybean in the southern United States. Analogs of CAP2b containing either a (Z)-alkene, cis-Pro or an (E)-alkene, trans-Pro isosteric component were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretion by Malpighian tubules isolated from A. hilare. The conformationally constrained trans-Pro analog demonstrated statistically significant antidiuretic activity, whereas the cis-Pro analog failed to elicit activity. The results are consistent with the adoption of a trans orientation for the Pro in CAP2b neuropeptides during interaction with receptors associated with the antidiuretic process in the stink bug. In addition, the results are further consistent with a theory of ligand-receptor coevolution between the CAP2b and pyrokinin/PBAN neuropeptide classes, both members of the '-PRXamide' superfamily. This work further identifies a scaffold with which to design mimetic CAP2b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting CAP2b-regulated diuretic/antidiuretic functions.


Subject(s)
Antidiuretic Agents/pharmacology , Heteroptera/physiology , Insect Proteins/pharmacology , Neuropeptides/pharmacology , Amino Acid Sequence , Animals , Antidiuretic Agents/chemistry , Evolution, Molecular , In Vitro Techniques , Insect Proteins/chemistry , Insect Proteins/physiology , Isomerism , Malpighian Tubules/drug effects , Malpighian Tubules/metabolism , Neuropeptides/chemistry , Neuropeptides/physiology , Proline/chemistry
14.
PLoS One ; 7(9): e44226, 2012.
Article in English | MEDLINE | ID: mdl-23028504

ABSTRACT

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.


Subject(s)
Cyclohexanes/chemistry , Ketones/chemistry , Peptidylprolyl Isomerase/chemistry , Ketones/antagonists & inhibitors , Molecular Conformation , Molecular Docking Simulation , Peptidylprolyl Isomerase/antagonists & inhibitors , Peptidylprolyl Isomerase/metabolism , Protein Binding
15.
ACS Chem Biol ; 7(8): 1462-70, 2012 Aug 17.
Article in English | MEDLINE | ID: mdl-22670809

ABSTRACT

The C-terminal domain (CTD) of eukaryotic RNA polymerase II is an essential regulator for RNA polymerase II-mediated transcription. It is composed of multiple repeats of a consensus sequence Tyr(1)Ser(2)Pro(3)Thr(4)Ser(5)Pro(6)Ser(7). CTD regulation of transcription is mediated by both phosphorylation of the serines and prolyl isomerization of the two prolines. Interestingly, the phosphorylation sites are typically close to prolines, and thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Experimental evidence of cross-talk between these two regulatory mechanisms has been elusive. Pin1 is a highly conserved phosphorylation-specific peptidyl-prolyl isomerase (PPIase) that recognizes the phospho-Ser/Thr (pSer/Thr)-Pro motif with CTD as one of its primary substrates in vivo. In the present study, we provide structural snapshots and kinetic evidence that support the concept of cross-talk between prolyl isomerization and phosphorylation. We determined the structures of Pin1 bound with two substrate isosteres that mimic peptides containing pSer/Thr-Pro motifs in cis or trans conformations. The results unequivocally demonstrate the utility of both cis- and trans-locked alkene isosteres as close geometric mimics of peptides bound to a protein target. Building on this result, we identified a specific case in which Pin1 differentially affects the rate of dephosphorylation catalyzed by two phosphatases (Scp1 and Ssu72) that target the same serine residue in the CTD heptad repeat but have different preferences for the isomerization state of the adjacent proline residue. These data exemplify for the first time how modulation of proline isomerization can kinetically impact signal transduction in transcription regulation.


Subject(s)
Peptidylprolyl Isomerase/chemistry , RNA Polymerase II/chemistry , Animals , Humans , Kinetics , Models, Chemical , Molecular Conformation , NIMA-Interacting Peptidylprolyl Isomerase , Phosphoric Monoester Hydrolases/chemistry , Phosphorylation , Phosphotransferases/chemistry , Proline/chemistry , Protein Binding , Protein Structure, Tertiary , Signal Transduction , Transcription, Genetic
16.
Nat Chem Biol ; 8(3): 262-9, 2012 Jan 22.
Article in English | MEDLINE | ID: mdl-22267120

ABSTRACT

Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked ß-N-acetyl-glucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2ß and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.


Subject(s)
Acetylglucosamine/metabolism , Casein Kinase II/metabolism , Animals , Casein Kinase II/biosynthesis , Casein Kinase II/chemistry , Cell Line , Humans , NIMA-Interacting Peptidylprolyl Isomerase , Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/metabolism , Phosphorylation , Rats , Serine/metabolism
17.
Biochemistry ; 50(44): 9545-50, 2011 Nov 08.
Article in English | MEDLINE | ID: mdl-21980916

ABSTRACT

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[CH(2)N]-Pro-2-(indol-3-yl)ethylamine, 1 [R = fluorenylmethoxycarbonyl (Fmoc)] and 2 (R = Ac), of Pin1 were synthesized and bioassayed. Inhibitor 1 had an IC(50) value of 6.3 µM, which is 4.5-fold better 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 IC(50) 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.


Subject(s)
Amides/chemistry , Enzyme Inhibitors/chemistry , Peptidylprolyl Isomerase/antagonists & inhibitors , Amides/chemical synthesis , Amides/metabolism , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Humans , NIMA-Interacting Peptidylprolyl Isomerase , Oxidation-Reduction , Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/metabolism , Protein Binding , Protein Conformation , Pyrrolidines/chemistry , Stereoisomerism
18.
Proc Natl Acad Sci U S A ; 108(30): 12289-94, 2011 Jul 26.
Article in English | MEDLINE | ID: mdl-21746900

ABSTRACT

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.


Subject(s)
Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/metabolism , Amino Acid Motifs , Binding, Competitive , Biophysical Phenomena , Catalytic Domain , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Hydrophobic and Hydrophilic Interactions , In Vitro Techniques , Models, Molecular , NIMA-Interacting Peptidylprolyl Isomerase , Nuclear Magnetic Resonance, Biomolecular , Peptidylprolyl Isomerase/antagonists & inhibitors , Peptidylprolyl Isomerase/genetics , Phosphorylation , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , Stereoisomerism , Substrate Specificity
19.
J Am Chem Soc ; 132(16): 5607-9, 2010 Apr 28.
Article in English | MEDLINE | ID: mdl-20356313

ABSTRACT

Drug design involves iterative ligand modifications. For flexible ligands, these modifications often entail restricting conformational flexibility. However, defining optimal restriction strategies can be challenging if the relationship between ligand flexibility and biological activity is unclear. Here, we describe an approach for ligand flexibility-activity studies using Nuclear Magnetic Resonance (NMR) spin relaxation. Specifically, we use (13)C relaxation dispersion measurements to compare site-specific changes in ligand flexibility for a series of related ligands that bind a common macromolecular receptor. The flexibility changes reflect conformational reorganization resulting from formation of the receptor-ligand complex. We demonstrate this approach on three structurally similar but flexibly differentiated ligands of human Pin1, a peptidyl-prolyl isomerase. The approach is able to map the ligand dynamics relevant for activity and expose changes in those dynamics caused by conformational locking. Thus, NMR flexibility-activity studies can provide information to guide strategic ligand rigidification. As such, they help establish an experimental basis for developing flexibility-activity relationships (FAR) to complement traditional structure-activity relationships (SAR) in molecular design.


Subject(s)
Oligopeptides/chemistry , Oligopeptides/metabolism , Peptidylprolyl Isomerase/metabolism , Amino Acid Sequence , Humans , Ligands , Magnetic Resonance Spectroscopy , NIMA-Interacting Peptidylprolyl Isomerase , Structure-Activity Relationship , Temperature
20.
Anal Biochem ; 402(1): 77-82, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20230769

ABSTRACT

Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr-Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr-Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr-Pro motif. An assay we call an enzyme-linked enzyme-binding assay (ELEBA) was developed to measure the K(d) of ligands that bind selectively to the WW domain. A ligand specific for the WW domain of Pin1 was covalently immobilized in a 96-well plate. Commercially available Pin1 conjugated to horseradish peroxidase was used for chemiluminescent detection of ligands that block the association of the WW domain with immobilized ligand. The peptide ligands were derived from the cell cycle regulatory phosphatase, Cdc25c, residues 45-50. The K(d) values for Fmoc-VPRpTPVGGGK-NH2 and Ac-VPRpTPV-NH2 were determined to be 36+/-4 and 110+/-30 microM, respectively. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain, even in the presence of the catalytic domain. This method may be applied to any dual specificity, multidomain protein.


Subject(s)
Enzyme Assays/methods , Peptides/metabolism , Peptidylprolyl Isomerase/metabolism , Amino Acid Sequence , Binding Sites , Horseradish Peroxidase/chemistry , Humans , Ligands , NIMA-Interacting Peptidylprolyl Isomerase , Peptides/chemistry , Peptidylprolyl Isomerase/chemistry , Protein Binding , Protein Structure, Tertiary , Sensitivity and Specificity
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