Human Pin1 Research Articles

Dissecting the Allosteric Fine-Tuning of Enzyme Catalysis.

Fully understanding the mechanism of allosteric regulation in biomolecules requires separating and examining all of the involved factors. In enzyme catalysis, allosteric effector binding shifts the structure and dynamics of the active site, leading to modified energetic (e.g., energy barrier) and dynamical (e.g., diffusion coefficient) factors underlying the catalyzed reaction rate. Such modifications can be subtle and dependent on the type of allosteric effector, representing a fine-tuning of protein function. The microscopic description of allosteric regulation at the level of function-dictating factors has prospective applications in fundamental and pharmaceutical sciences, which is, however, largely missing so far. Here, we characterize the allosteric fine-tuning of enzyme catalysis, using human Pin1 as an example, by performing more than half-millisecond all-atom molecular dynamics simulations. Changes of reaction kinetics and the dictating factors, including the free energy surface along the reaction coordinate and the diffusion coefficient of the reaction dynamics, under various enzyme and allosteric effector binding conditions are examined. Our results suggest equal importance of the energetic and dynamical factors, both of which can be modulated allosterically, and the combined effect determines the final allosteric output. We also reveal the potential dynamic basis for allosteric modulation using an advanced statistical technique to detect function-related conformational dynamics. Methods developed in this work can be applied to other allosteric systems.

Cost-effective selective deuteration of aromatic amino acid residues produces long-lived solution 1H NMR magnetization in proteins

Solution NMR studies of large proteins are hampered by rapid signal decay due to short-range dipolar 1H–1H and 1H–13C interactions. These are attenuated by rapid rotation in methyl groups and by deuteration (2H), so selective 1H,13C-isotope labelling of methyl groups in otherwise perdeuterated proteins, combined with methyl transverse relaxation optimized spectroscopy (methyl-TROSY), is now standard for solution NMR of large protein systems > 25 kDa. For non-methyl positions, long-lived magnetization can be introduced as isolated 1H–12C groups. We have developed a cost-effective chemical synthesis for producing selectively deuterated phenylpyruvate and hydroxyphenylpyruvate. Feeding these amino acid precursors to E. coli in D2O, along with selectively deuterated anthranilate and unlabeled histidine, results in isolated and long-lived 1H magnetization in the aromatic rings of Phe (HD, HZ), Tyr (HD), Trp (HH2, HE3) and His (HD2 and HE1). We are additionally able to obtain stereoselective deuteration of Asp, Asn, and Lys amino acid residues using unlabeled glucose and fumarate as carbon sources and oxalate and malonate as metabolic inhibitors. Combining these approaches produces isolated 1H–12C groups in Phe, Tyr, Trp, His, Asp, Asn, and Lys in a perdeuterated background, which is compatible with standard 1H–13C labeling of methyl groups in Ala, Ile, Leu, Val, Thr, Met. We show that isotope labeling of Ala is improved using the transaminase inhibitor L-cycloserine, and labeling of Thr is improved through addition of Cys and Met, which are known inhibitors of homoserine dehydrogenase. We demonstrate the creation of long-lived 1H NMR signals in most amino acid residues using our model system, the WW domain of human Pin1, as well as the bacterial outer membrane protein PagP.

A Redox-Sensitive Cysteine Is Required for PIN1At Function.

Parvulins are ubiquitous peptidyl-prolyl isomerases (PPIases) required for protein folding and regulation. Among parvulin members, Arabidopsis PIN1At, human PIN1, and yeast ESS1 share a conserved cysteine residue but differ by the presence of an N-terminal WW domain, absent in PIN1At. In this study, we have explored whether the cysteine residue of Arabidopsis PIN1At is involved in catalysis and subject to oxidative modifications. From the functional complementation of yeast ess1 mutant, we concluded that the cysteine at position 69 is mandatory for PIN1At function in vivo, unless being replaced by an Asp which is found in a few parvulin members. This result correlates with a decrease of the in vitro PPIase activity of non-functional PIN1At cysteinic variants. A decrease of PIN1At activity was observed upon H2O2 treatment. The in vitro oxidation of cysteine 69, which has an acidic pKa value of 4.9, leads to the formation of covalent dimers that are reduced by thioredoxins, or to sulfinic or sulfonic acid forms at higher H2O2 excess. These investigations highlight the importance of the sole cysteine residue of PIN1At for activity. The reversible formation of an intermolecular disulfide bond might constitute a protective or regulatory mechanism under oxidizing conditions.

PEGylation Increases the Strength of a Nearby NH-π Hydrogen Bond in the WW Domain

Here we show that an NH-π interaction between a highly conserved Asn and a nearby Trp stabilizes the WW domain of the human protein Pin1. The strength of this NH-π interaction depends on the structure of the arene, with NH-π interactions involving Trp or naphthylalanine being substantially more stabilizing than those involving Tyr or Phe. Calculations suggest arene size and polarizability are key structural determinants of NH-π interaction strength. Methylation or PEGylation of the Asn side-chain amide nitrogen each strengthens the associated NH-π interaction, though likely for different reasons. We hypothesize that methylation introduces steric clashes that destabilize conformations in which the NH-π interaction is not possible, whereas PEGylation strengthens the NH-π interaction via localized desolvation of the protein surface.

A Meat-Derived Lactic Acid Bacteria, Lactobacillus plantarum IIA, Expresses a Functional Parvulin-Like Protein with Unique Structural Property

The genome sequence of a Lactic Acid Bacterium (LAB) Lactobacillus plantarum IIA contains a single gene encoding a parvulin-like protein (Par-LpIIA). This protein belongs to Peptidyl Prolyl cis-trans Isomerase (PPIase) family proteins that catalyze a slow cis-trans isomerization of cis prolyl bond during protein folding. This study aims to provide molecular and biochemical evidences of the existence of Par-LpIIA in L. plantarum IIA and have an insight into its structural properties. The result showed that the gene encoding Par-LpIIA was successfully amplified using specific primers yielding a ~900 bp amplicon indicating that the gene indeed exists in its genomic DNA. BLAST analysis confirmed that the protein is a rotamase of parvulin-like protein. Further biochemical analysis demonstrated that cell lysate of L. plantarum IIA-1A5 exhibited remarkable PPIase activity towards peptide substrate and ability to accelerate the refolding of RNase T1, with the catalytic efficiency (kcat/KM) of 1.9 and 0.02 µM-1 s-1, respectively. A specific inhibitor clearly inhibited the PPIase activity for parvulin-like protein with IC50 of 230 nM confirming that the protein encoded by Par-LpIIA gene is a parvulin-like protein and expressed in an active form. Further, the three-dimensional model of Par-LpIIA showed that this protein consists of two domains of a homolog WW domain and PPIase domain with a unique active site configuration compared to human Pin1. Altogether, we then proposed the possible roles of this protein for L. plantarum IIA.

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.

Design, synthesis and biological evaluation of novel thiazole-based derivatives as human Pin1 inhibitors

Pin1 is a peptidyl prolyl cis-trans isomerase (PPIase) and inhibiting Pin1 is a potential way for discovering anti-tumor agents. With an aim to find potent Pin1 inhibitors with a novel scaffold, a series of thiazole derivatives with an alicyclic heterocycles on the 2-position were designed, synthesized and tested against human Pin1. Compound 9p bearing a 2-oxa-6-azaspiro [3,3] heptane moiety on the thiazole scaffold was identified as the most potent Pin1 inhibitor of this series with an IC50 value of 0.95 μM. The structure-activity relationship (SAR) and molecular modeling study indicated that introducing an alicyclic ring with an H-bond acceptor would be a viable way to improve the binding affinity.

Reducing the measurement time of exact NOEs by non-uniform sampling.

We have previously reported on the measurement of exact NOEs (eNOEs), which yield a wealth of additional information in comparison to conventional NOEs. We have used these eNOEs in a variety of applications, including calculating high-resolution structures of proteins and RNA molecules. The collection of eNOEs is challenging, however, due to the need to measure a NOESY buildup series consisting of typically four NOESY spectra with varying mixing times in a single measurement session. While the 2D version can be completed in a few days, a fully sampled 3D-NOESY buildup series can take 10days or more to acquire. This can be both expensive as well as problematic in the case of samples that are not stable over such a long period of time. One potential method to significantly decrease the required measurement time of eNOEs is to use non-uniform sampling (NUS) to decrease the number of points measured in the indirect dimensions. The effect of NUS on the extremely tight distance restraints extracted from eNOEs may be very pronounced. Therefore, we investigated the fidelity of eNOEs measured from three test cases at decreasing NUS densities: the 18.4kDa protein human Pin1, the 4.1kDa WW domain of Pin1 (both in 3D), and a 4.6kDa 14mer RNA UUCG tetraloop (2D). Our results show that NUS imparted negligible error on the eNOE distances derived from good quality data down to 10% sampling for all three cases, but there is a noticeable decrease in the eNOE yield that is dependent upon the underlying sparsity, and thus complexity, of the sample. For Pin1, this transition occurred at roughly 40% while for the WW domain and the UUCG tetraloop it occurred at lower NUS densities of 20% and 10%, respectively. We rationalized these numbers through reconstruction simulations under various conditions. The extent of this loss depends upon the number of scans taken as well as the number of peaks to be reconstructed. Based on these findings, we have created guidelines for choosing an optimal NUS density depending on the number of peaks needed to be reconstructed in the densest region of a 2D or 3D NOESY spectrum.

Acidic-basic properties of arginine-rich peptide fragments derived from the human Pin1 protein

The effects of arginine residues in peptide sequences on their specific acid-base properties were investigated. Two short original fragments of the human Pin1 WW domain (hPin1 14–24; hPin1 15–23) and one single point mutation system (the original charged Lys14 and Tyr24 residues were replaced with nonpolar alanine residues) derived from hPin1 were considered. The amino acid composition controls to a large extent the process of folding and thus regulates the functions of the entire system. The specific behavior of arginine residues presented in the investigated peptides in a solution is directly reflected in their abnormally low values of pKa as compared to those of the reference compound. This phenomenon seems to result from the formation of aggregates within the system by the guanidine forms present in the Arg sidechains. The acid-base properties of the investigated peptides show that under physiological conditions, the charge of these systems is close to 2. Summarizing, the analysis of interactions between peptides and biologically important structures and membranes should consider cationic forms of peptides.

Solution Structure Studies of Ess1 Interactions with the RNAPII CTD Suggest a Dual Binding Mechanism that Differs from that of Human Pin1

Solution Structure Studies of Ess1 Interactions with the RNAPII CTD Suggest a Dual Binding Mechanism that Differs from that of Human Pin1

Targeting PIN1 exerts potent antitumor activity in pancreatic ductal carcinoma via inhibiting tumor metastasis.

The human prolyl isomerase PIN1, best known for its association with carcinogenesis, has recently been indicated in the disease of pancreatic ductal adenocarcinoma (PDAC). However, the functions of PIN1 and the feasibility of targeting PIN1 in PDAC remain elusive. For this purpose, we examined the expression of PIN1 in cancer, related paracarcinoma and metastatic cancer tissues by immunohistochemistry and analyzed the associations with the pathogenesis of PDAC in 173 patients. The functional roles of PIN1 in PDAC were explored in vitro and in vivo using both genetic and chemical PIN1 inhibition. We showed that PIN1 was upregulated in pancreatic cancer and metastatic tissues. High PIN1 expression is significantly association with poor clinicopathological features and shorter overall survival and disease‐free survival. Further stratified analysis showed that PIN1 phenotypes refined prognostication in PDAC. Inhibition of PIN1 expression with RNA interference or with all trans retinoic acid decreased not only the growth but also the migration and invasion of PDAC cells through regulating the key molecules of multiple cancer‐driving pathways, simultaneously resulting in cell cycle arrest and mesenchymal‐epithelial transition in vitro. Furthermore, genetic and chemical PIN1 ablation showed dramatic inhibition of the tumorigenesis and metastatic spread and then reduced the tumor burden in vivo. We provided further evidence for the use of PIN1 as a promising therapeutic target in PDAC. Genetic and chemical PIN1 ablation exerted potent antitumor effects through blocking multiple cancer‐driving pathways in PDAC. More potent and specific PIN1 targeted inhibitors could be exploited to treat this aggressive cancer.

Using Histone H1 Derived Peptides to Investigate Binding Affinity and Inter-Domain Dynamics in Human Pin1

Pin1 is an essential Peptidyl-prolyl isomerase (PPIase) that catalyzes cis-trans prolyl isomerization in proteins containing pSer/pThr-Pro motifs. It has an N-terminal WW domain that targets pSer/Thr-Pro motifs and a C-terminal PPIase domain that catalyzes isomerization. In this study, we report a novel series of chimeric peptides that has been designed to optimize binding across the inter-domain interface of Pin1. The peptide series was designed based on the binding site of the Pin1 and optimal peptide sequences analyzed in earlier studies. Specifically, we started from a sequence derived from the human Histone H1.4 sequence (KATGAApTPKKSAKW). Then, we designed a series of chimeric peptides based on this H1.4 sequence. NMR titrations were performed for each peptide using both full-length Pin1 as well as the WW domain alone. An analysis of KD values reveals complexity in the energetics of interaction between the peptide substrate, the PPIase domain, and the WW domain. 15N relaxation and residual dipolar couplings (RDCs) were used to monitor the degree to which peptide binding induced inter-domain interactions. When combined with molecular simulations, our results suggest a structural basis for how substrate binding can alter the inter-domain dynamics. Finally, we investigated whether our chimeric sequences could alter catalysis (kex) using 1H-1H EXSY NMR experiments. Interestingly, no relationship was found between kex and either peptide affinity or inter-domain interaction, suggesting a lack of allosteric control for this series of peptides. Thus, while our results suggest that peptide binding can alter the interaction between the PPIase and WW domains, altering the inter-domain interaction by itself does not appear to modulate catalysis in the PPIase domain.

Backbone and side-chain chemical shift assignments of full-length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery.

Pin1 is a human peptidyl-prolyl cis-trans isomerase important for the regulation of phosphoproteins that are implicated in many diseases including cancer and Alzheimer's. Further biophysical study of Pin1 will elucidate the importance of the two-domain system to regulate its own activity. Here, we report near-complete backbone and side-chain 1H, 13C and 15N NMR chemical shift assignments of full-length, apo Pin1 for the purpose of studying interdomain allostery and dynamics.

Regulation of the Minichromosome Maintenance Protein 3 (MCM3) Chromatin Binding by the Prolyl Isomerase Pin1

Human Pin1 is a peptidyl prolyl cis/trans isomerase with a unique preference for phosphorylated Ser/Thr-Pro substrate motifs.Here we report that MCM3 (minichromosome maintenance complex component 3) is a novel target of Pin1. MCM3 interacts directly with the WW domain of Pin1. Proline-directed phosphorylation of MCM3 at S112 and T722 are crucial for the interaction with Pin1. MCM3 as a subunit of the minichromosome maintenance heterocomplex MCM2–7 is part of the pre-replication complex responsible for replication licensing and is implicated in the formation of the replicative helicase during progression of replication. Our data suggest that Pin1 coordinates phosphorylation-dependently MCM3 loading onto chromatin and its unloading from chromatin, thereby mediating S phase control.

Enhanced Sampling of Interdomain Motion Using Map-Restrained Langevin Dynamics and NMR: Application to Pin1

Many signaling proteins consist of globular domains connected by flexible linkers that allow for substantial domain motion. Because these domains often serve as complementary functional modules, the possibility of functionally important domain motions arises. To explore this possibility, we require knowledge of the ensemble of protein conformations sampled by interdomain motion.Measurements of NMR residual dipolar couplings (RDCs) of backbone HN bonds offer a per-residue characterization of interdomain dynamics, as the couplings are sensitive to domain orientation. A challenge in reaching this potential is the need to interpret the RDCs as averages over dynamic ensembles of domain conformations.Here, we address this challenge by introducing an efficient protocol for generating conformational ensembles appropriate for flexible, multi-domain proteins. The protocol uses map-restrained self-guided Langevin dynamics simulations to promote collective, interdomain motion while restraining the internal domain motion to near rigidity. Critically, the simulations retain an all-atom description for facile inclusion of site-specific NMR RDC restraints. The result is the rapid generation of conformational ensembles consistent with the RDC data.We illustrate this protocol on human Pin1, a two-domain peptidyl-prolyl isomerase relevant for cancer and Alzheimer's disease. The results include the ensemble of domain orientations sampled by Pin1, as well as those of a dysfunctional variant, I28A-Pin1. The differences between the ensembles corroborate our previous spin relaxation results that showed weakened interdomain contact in the I28A variant relative to wild type. Our protocol extends our abilities to explore the functional significance of protein domain motions.

Synthesis and biological evaluation of pyrimidine derivatives as novel human Pin1 inhibitors

Pin1 (Protein interacting with NIMA1) is a cis–trans isomerase and promotes the amide bond rotation of phosphoSer/Thr-Pro motifs in its substrates. Inhibition of Pin1 might be a novel strategy for developing anticancer agents. Herein, a series of pyrimidine derivatives were synthesized and their Pin1 inhibitory activities were evaluated. Among them, four compounds (2a, 2f, 2h and 2l) displayed potent inhibitory activities against Pin1 with IC50 values lower than 3 µM. This series of pyrimidine-based inhibitors presented time-dependent inhibition against Pin1. The structure–activity relationships on the 2-, 4- and 5-positions of the pyrimidine ring were analyzed in details, which would facilitate further exploration of new Pin1 inhibitors.

Hinge-Shift Mechanism Modulates Allosteric Regulations in Human Pin1.

Allostery, which is regulation from distant sites, plays a major role in biology. While traditional allostery is described in terms of conformational change upon ligand binding as an underlying principle, it is possible to have allosteric regulations without significant conformational change through modulating the conformational dynamics by altering the local effective elastic modulus of the protein upon ligand binding. Pin1 utilizes this dynamic allostery to regulate its function. It is a modular protein containing a WW domain and a larger peptidyl prolyl isomerase domain (PPIase) that isomerizes phosphoserine/threonine-proline (pS/TP) motifs. The WW domain serves as a docking module, whereas catalysis solely takes place within the PPIase domain. Here, we analyze the change in the dynamic flexibility profile of the PPIase domain upon ligand binding to the WW domain. Substrate binding to the WW domain induces the formation of a new rigid hinge site around the interface of the two domains and loosens the flexibility of a rigid site existing in the Apo form around the catalytic site. This hinge-shift mechanism enhances the dynamic coupling of the catalytic positions with the PPIase domain, where the rest of the domain can cooperatively respond to the local conformational changes around the catalytic site, leading to an increase in catalytic efficiency.

Structure and function of the human parvulins Pin1 and Par14/17.

Parvulins belong to the family of peptidyl-prolyl cis/trans isomerases (PPIases) assisting in protein folding and in regulating the function of a broad variety of proteins in all branches of life. The human representatives Pin1 and Par14/17 are directly involved in processes influencing cellular maintenance and cell fate decisions such as cell-cycle progression, metabolic pathways and ribosome biogenesis. This review on human parvulins summarizes the current knowledge of these enzymes and intends to oppose the well-studied Pin1 to its less well-examined homolog human Par14/17 with respect to structure, catalytic and cellular function.

Stereoselective Deuteration in Aspartate, Asparagine, Lysine, and Methionine Amino Acid Residues Using Fumarate as a Carbon Source for Escherichia coli in D2O.

Perdeuteration with selective 1H,13C-enrichment of methyl groups has enabled solution NMR studies of large (>30 kDa) protein systems. However, we propose that for all non-methyl positions, only magnetization originating from 1H-12C groups is sufficiently long-lived, and it can be transferred via through-space NOEs to slowly relaxing 1H-15N or 1H-13C methyl groups to achieve multidimensional solution NMR. We demonstrate stereoselective 1H,12C-labeling by adding relatively inexpensive unlabeled carbon sources to Escherichia coli growth media in D2O. Using our model system, a mutant WW domain from human Pin1, we compare deuteration patterns in 19 amino acids (all except cysteine). Protein grown using glucose as the sole carbon source had high levels of protonation in aromatic rings and the Hβ positions of serine and tryptophan. In contrast, using our FROMP media (fumarate, rhamnose, oxalate, malonate, pyruvate), stereoselective protonation of Hβ2 with deuteration at Hα and Hβ3 was achieved in Asp, Asn, Lys, and Met residues. In solution NMR, stereospecific chemical shift assignments for Hβ are typically obtained in conjunction with χ1 dihedral angle determinations using 3-bond J-coupling (3JN-Hβ, 3JCO-Hβ, 3JHα-Hβ) experiments. However, due to motional averaging, the assumption of a pure rotameric state can yield incorrect χ1 dihedral angles with incorrect stereospecific assignments. This was the case for three residues in the Pin1 WW domain (Lys28, Met30, and Asn44). Thus, stereoselective 1H,12C-labeling will be useful not only for NMR studies of large protein systems, but also for determining side chain rotamers and dynamics in any protein system.

Using Cooperatively Folded Peptides To Measure Interaction Energies and Conformational Propensities.

The rates and equilibria of the folding of biopolymers are determined by the conformational preferences of the subunits that make up the sequence of the biopolymer and by the interactions that are formed in the folded state in aqueous solution. Because of the centrality of these processes to life, quantifying conformational propensities and interaction strengths is vitally important to understanding biology. In this Account, we describe our use of peptide model systems that fold cooperatively yet are small enough to be chemically synthesized to measure such quantities. The necessary measurements are made by perturbing an interaction or conformation of interest by mutation and measuring the difference between the folding free energies of the wild type (in which the interaction or conformation is undisturbed) and the mutant model peptides (in which the interaction has been eliminated or the conformational propensities modified). With the proper controls and provided that the peptide model system in question folds via a two-state process, these folding free energy differences can be accurate measures of interaction strengths or conformational propensities. This method has the advantage of having high sensitivity and high dynamic range because the energies of interest are coupled to folding free energies, which can be measured with precisions on the order of a few tenths of a kilocalorie by well-established biophysical methods, like chaotrope or thermal denaturation studies monitored by fluorescence or circular dichroism. In addition, because the model peptides can be chemically synthesized, the full arsenal of natural and unnatural amino acids can be used to tune perturbations to be as drastic or subtle as desired. This feature is particularly noteworthy because it enables the use of analytical tools developed for physical organic chemistry, especially linear free energy relationships, to decompose interaction energies into their component parts to obtain a deeper understanding of the forces that drive interactions in biopolymers. We have used this approach, primarily with the WW domain derived from the human Pin1 protein as our model system, to assess hydrogen bond strengths (especially those formed by backbone amides), the dependence of hydrogen bond strengths on the environment in which they form, β-turn propensities of both natural sequences and small molecule β-turn mimics, and the energetics of carbohydrate-protein interactions. In each case, the combination of synthetic accessibility, the ease of measuring folding energies, and the robustness of the structure of the Pin1 WW domain to mutation enabled us to obtain incisive measurements of quantities that have been challenging to measure by other methods.