A Reduced-Amide Inhibitor of Pin1 Binds in a Conformation Resembling a Twisted-Amide Transition State

Xu, Guanghui; Zhang, Yan; Mercedes-Camacho, Ana Y.; Etzkorn, Felicia A.

doi:10.1021/bi201055c

Cited by 30 publications

(65 citation statements)

References 40 publications

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“…We anticipated that the ketones would be poor inhibitors, while the reduced amides, as twisted-amide analogues, would fare better. Indeed, the reduced amide 3 is a better Pin1 inhibitor than a similarly substituted substrate analogue ( Z )-alkene isostere 5 (Figure 2) [13], [27]. Our crystal structure of reduced amide 4 bound to the Pin1 catalytic site adopted a trans -pyrrolidine conformation, supporting the twisted-amide mechanism [27].…”

Section: Introductionsupporting

confidence: 53%

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

Xu¹,

Slebodnick²,

Etzkorn

2012

PLoS ONE

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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.

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Section: Introductionsupporting

confidence: 53%

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

Xu¹,

Slebodnick²,

Etzkorn

2012

PLoS ONE

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“…The crystal structure of a reduced amide−Pin1 complex (PDB entry 3NTP), where the structure is considered to mimic the transition state complex with pSer-Pro, showed that the basic triad residues form hydrogen bonds to the phosphate moiety in the substrate. 19 The side chain reorientation of the basic triad in the C113D mutant may explain the severely reduced capacity for binding to the phosphorylated substrate ( Figure 1B).…”

Section: ■ Resultsmentioning

confidence: 99%

“…18 The crystal structure of Pin1 with a reduced amide inhibitor bound in the active site showed the compound structure was close to the twisted amide, which is proposed to exist in a transition state. 19 Molecular dynamics simulations gave other insights into the cis−trans isomerization process. Hamelberg and co-workers applied molecular dynamics simulations to sample the entire conformational ensemble of the Pin1 substrate complex, with the corresponding relative binding free energies, during the cis− trans conversion of the substrate.…”

Section: * S Supporting Informationmentioning

confidence: 99%

The C113D Mutation in Human Pin1 Causes Allosteric Structural Changes in the Phosphate Binding Pocket of the PPIase Domain through the Tug of War in the Dual-Histidine Motif

Tochio

Wang

et al. 2014

Biochemistry

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Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes specifically the pSer/pThr-Pro motif. The cis-trans isomerization mechanism has been studied by various approaches, including X-ray crystallography, site-directed mutagenesis, and the kinetic isotope effect on isomerization. However, a complete picture of the reaction mechanism remains elusive. On the basis of the X-ray structure of Pin1, residue C113 was proposed to play a nucleophile attacker to catalyze the isomerization. The controversial result that the C113D Pin1 mutant retains the activity, albeit at a reduced level, challenges the importance of C113 as a catalyst. To facilitate our understanding of the Pin1 isomerization process, we compared the structures and dynamics of the wild type with those of the C113D mutant Pin1 PPIase domains (residues 51-163). We found the C113D mutation disturbed the hydrogen bonds between the conserved histidine residues, H59 and H157 ("dual-histidine motif"); H59 imidazole forms a stable hydrogen bond to H157 in the wild type, whereas it has a strong hydrogen bond to D113 with weakened bonding to H157 in the C113D mutant. The C113D mutation unbalanced the hydrogen bonding tug of war for H59 between C113/D113 and H157 and destabilized the catalytic site structure, which eventually resulted in an altered conformation of the basic triad (K63, R68, and R69) that binds to the phosphate group in a substrate. The change in the basic triad structure could explain the severely weakened substrate binding ability of the C113D mutant. Overall, this work demonstrated that C113 plays a role in keeping the catalytic site in an active fold, which has never before been described.

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“…41, 42 In addition, the flexibility of the interactions between the phosphate group and the positive triad during the rotation permits transition-state stabilization. 36 …”

Section: Discussionmentioning

confidence: 99%

Structural and Kinetic Analysis of Prolyl-isomerization/Phosphorylation Cross-Talk in the CTD Code

et al. 2012

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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 Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. Ser2 and Ser5 are the major phosphorylation sites in vivo while Pro3 and Pro6 can adopt either cis or trans conformations. CTD regulation of transcription is mediated both by phosphorylation of the serines and prolyl isomerization of the two prolines. Interestingly, the phosphorylation sites are typically close to prolines, 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 that 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.

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A Reduced-Amide Inhibitor of Pin1 Binds in a Conformation Resembling a Twisted-Amide Transition State

Cited by 30 publications

References 40 publications

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

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

The C113D Mutation in Human Pin1 Causes Allosteric Structural Changes in the Phosphate Binding Pocket of the PPIase Domain through the Tug of War in the Dual-Histidine Motif

Structural and Kinetic Analysis of Prolyl-isomerization/Phosphorylation Cross-Talk in the CTD Code

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