Biochemical characterization of the Eya and PP2A-B55α interaction.

The eyes absent (Eya) proteins were first identified as co-activators of the six homeobox family of transcription factors and are critical in embryonic development. These proteins are also re-expressed in cancers after development is complete, where they drive tumor progression. We have previously shown that the Eya3 N-terminal domain (NTD) contains Ser/Thr phosphatase activity through an interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme and that this interaction increases the half-life of Myc through pT58 dephosphorylation. Here, we showed that Eya3 directly interacted with the NTD of Myc, recruiting PP2A-B55α to Myc. We also showed that Eya3 increased the Ser/Thr phosphatase activity of PP2A-B55α but not PP2A-B56α. Furthermore, we demonstrated that the NTD (∼250 amino acids) of Eya3 was completely disordered, and it used a 38-residue segment to interact with B55α. In addition, knockdown and phosphoproteomic analyses demonstrated that Eya3 and B55α affected highly similar phosphosite motifs with a preference for Ser/Thr followed by Pro, consistent with Eya3's apparent Ser/Thr phosphatase activity being mediated through its interaction with PP2A-B55α. Intriguingly, mutating this Pro to other amino acids in a Myc peptide dramatically increased dephosphorylation by PP2A. Not surprisingly, MycP59A, a naturally occurring mutation hotspot in several cancers, enhanced Eya3-PP2A-B55α-mediated dephosphorylation of pT58 on Myc, leading to increased Myc stability and cell proliferation, underscoring the critical role of this phosphosite in regulating Myc stability.

Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126.

To date, there are very limited reports on sequence analysis and structure-based molecular modeling of phosphatases produced by probiotic bacteria. Therefore, a novel protein tyrosine-like phosphatase was characterized from L. helveticus 2126 in this study. The purified bacterial phosphatase was subjected to mass spectrometric analysis, and the identity of constructed sequence was analyzed using peptide mass fingerprint. The 3-D structure of protein was elucidated using homology modeling, while its stability was assessed using Ramachandran plot, VERIFY 3D, and PROCHECK. The bacterium produced an extracellular phosphatase of zone diameter 15 ± 0.8 mm on screening medium within 24 h of incubation. This bacterial phosphatase was highly specific towards sodium phytate as it yielded the lowest Km value of 299.50 ± 4.95 µM compared to other phosphorylated substrates. The activity was effectively stimulated in the presence of zinc, magnesium, and manganese ions thereby showing its PTP-like behavior. The phosphatase showed a molecular mass of 43 kDa, and the corresponding M/Z ratio data yielded 46% query coverage to Bacillus subtilis (3QY7). This showed a 61.1% sequence similarity to Ligilactobacillus ruminis (WP_046923835.1). The final sequence construct based on these bacteria showed a conserved motif "HCHILPGIDD" in their active site. In addition, homology modeling showed a distorted Tim barrel structure with a trinuclear metal center. The final model after energy minimization showed 90.9% of the residues in the favorable region of Ramachandran's plot. This structural information can be used in genetic engineering for improving the overall stability and catalytic efficiency of probiotic bacterial phosphatases.

The PTP family photo album.

Protein tyrosine phosphatases (PTPs) are central players in many biological processes. In this issue, Barr et al. (2009) analyze 22 different PTP structures to define their common and unique features. This effort provides key insights into the regulation of PTP activity that could lead to the development of new therapeutics.

Large-scale structural analysis of the classical human protein tyrosine phosphatome.

Protein tyrosine phosphatases (PTPs) play a critical role in regulating cellular functions by selectively dephosphorylating their substrates. Here we present 22 human PTP crystal structures that, together with prior structural knowledge, enable a comprehensive analysis of the classical PTP family. Despite their largely conserved fold, surface properties of PTPs are strikingly diverse. A potential secondary substrate-binding pocket is frequently found in phosphatases, and this has implications for both substrate recognition and development of selective inhibitors. Structural comparison identified four diverse catalytic loop (WPD) conformations and suggested a mechanism for loop closure. Enzymatic assays revealed vast differences in PTP catalytic activity and identified PTPD1, PTPD2, and HDPTP as catalytically inert protein phosphatases. We propose a "head-to-toe" dimerization model for RPTPgamma/zeta that is distinct from the "inhibitory wedge" model and that provides a molecular basis for inhibitory regulation. This phosphatome resource gives an expanded insight into intrafamily PTP diversity, catalytic activity, substrate recognition, and autoregulatory self-association.

In-silico identification of Tyr232 in AMPKα2 as a dephosphorylation site for the protein tyrosine phosphatase PTP-PEST.

The AMP-activated protein kinase (AMPK) is known to be activated by the protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) under hypoxic conditions. This activation is mediated by tyrosine dephosphorylation of the AMPKα subunit. However, the identity of the phosphotyrosine residues that PTP-PEST dephosphorylates remains unknown. In this study, we first predicted the structure of the complex of the AMPKα2 subunit and PTP-PEST catalytic domain using bioinformatics tools and further confirmed the stability of the complex using molecular dynamics simulations. Evaluation of the protein-protein interfaces indicated that residue Tyr232 is the most likely dephosphorylation site on AMPKα2. In addition, we explored the effect of phosphorylation of PTP-PEST residue Tyr64 on the stability of the complex. Phosphorylation of the highly conserved Tyr64, an interface residue, enhances the stability of the complex via the rearrangement of a network of electrostatic interactions in conjunction with conformational changes in the catalytic WPD loop. We generated a phosphomimetic (PTP-PEST-Y64D) mutant and used co-immunoprecipitation to study the effect of PTP-PEST phosphorylation on AMPKα2 binding. The mutant exhibited an increased affinity for AMPKα2 and corroborated the in-silico predictions. Together, our findings present a plausible structural basis of AMPK regulation by PTP-PEST and show how phosphorylation of PTP-PEST affects its interaction with AMPKα2.

Mapping the Chemical Space of Active-Site Targeted Covalent Ligands for Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) are an important class of enzymes that modulate essential cellular processes through protein dephosphorylation and are dysregulated in various disease states. There is demand for new compounds that target the active sites of these enzymes, for use as chemical tools to dissect their biological roles or as leads for the development of new therapeutics. In this study, we explore an array of electrophiles and fragment scaffolds to investigate the required chemical parameters for covalent inhibition of tyrosine phosphatases. Our analysis juxtaposes the intrinsic electrophilicity of these compounds with their potency against several classical PTPs, revealing chemotypes that inhibit tyrosine phosphatases while minimizing excessive, potentially non-specific reactivity. We also assess sequence divergence at key residues in PTPs to explain their differential susceptibility to covalent inhibition. We anticipate that our study will inspire new strategies to develop covalent probes and inhibitors for tyrosine phosphatases.

Real-time observation of ligand-induced allosteric transitions in a PDZ domain.

While allostery is of paramount importance for protein regulation, the underlying dynamical process of ligand (un)binding at one site, resulting time evolution of the protein structure, and change of the binding affinity at a remote site are not well understood. Here the ligand-induced conformational transition in a widely studied model system of allostery, the PDZ2 domain, is investigated by transient infrared spectroscopy accompanied by molecular dynamics simulations. To this end, an azobenzene-derived photoswitch is linked to a peptide ligand in a way that its binding affinity to the PDZ2 domain changes upon switching, thus initiating an allosteric transition in the PDZ2 domain protein. The subsequent response of the protein, covering four decades of time, ranging from ∼1 ns to ∼µs, can be rationalized by a remodeling of its rugged free-energy landscape, with very subtle shifts in the populations of a small number of structurally well-defined states. It is proposed that structurally and dynamically driven allostery, often discussed as limiting scenarios of allosteric communication, actually go hand-in-hand, allowing the protein to adapt its free-energy landscape to incoming signals.

Recent advances in the development of allosteric protein tyrosine phosphatase inhibitors for drug discovery.

Protein tyrosine phosphatases (PTPs) superfamily catalyzes tyrosine de-phosphorylation which affects a myriad of cellular processes. Imbalance in signal pathways mediated by PTPs has been associated with development of many human diseases including cancer, metabolic, and immunological diseases. Several compelling evidence suggest that many members of PTP family are novel therapeutic targets. However, the clinical development of conventional PTP-based active-site inhibitors originally was hampered by the poor selectivity and pharmacokinetic properties. In this regard, PTPs has been widely dismissed as "undruggable." Nonetheless, allosteric modulation has become increasingly an influential and alternative approach that can be exploited for drug development against PTPs. Unlike active-site inhibitors, allosteric inhibitors exhibit a remarkable target-selectivity, drug-likeness, potency, and in vivo activity. Intriguingly, there has been a high interest in novel allosteric PTPs inhibitors within the last years. In this review, we focus on the recent advances of allosteric inhibitors that have been explored in drug discovery and have shown an excellent result in the development of PTPs-based therapeutics. A special emphasis is placed on the structure-activity relationship and molecular mechanistic studies illustrating applications in chemical biology and medicinal chemistry.

The STEP61 interactome reveals subunit-specific AMPA receptor binding and synaptic regulation.

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific protein phosphatase that regulates a variety of synaptic proteins, including NMDA receptors (NAMDRs). To better understand STEP's effect on other receptors, we used mass spectrometry to identify the STEP61 interactome. We identified a number of known interactors, but also ones including the GluA2 subunit of AMPA receptors (AMPARs). We show that STEP61 binds to the C termini of GluA2 and GluA3 as well as endogenous AMPARs in hippocampus. The synaptic expression of GluA2 and GluA3 is increased in STEP-KO mouse brain, and STEP knockdown in hippocampal slices increases AMPAR-mediated synaptic currents. Interestingly, STEP61 overexpression reduces the synaptic expression and synaptic currents of both AMPARs and NMDARs. Furthermore, STEP61 regulation of synaptic AMPARs is mediated by lysosomal degradation. Thus, we report a comprehensive list of STEP61 binding partners, including AMPARs, and reveal a central role for STEP61 in differentially organizing synaptic AMPARs and NMDARs.

RPTPα phosphatase activity is allosterically regulated by the membrane-distal catalytic domain.

Receptor-type protein tyrosine phosphatase α (RPTPα) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. The domain structure of RPTPs comprises an extracellular region, a transmembrane helix, and two tandem intracellular catalytic domains referred to as D1 and D2. The D2 domain of RPTPs is believed to mostly play a regulatory function; however, no regulatory model has been established for RPTPα-D2 or other RPTP-D2 domains. Here, we solved the 1.8 Å resolution crystal structure of the cytoplasmic region of RPTPα, encompassing D1 and D2, trapped in a conformation that revealed a possible mechanism through which D2 can allosterically inhibit D1 activity. Using a D2-truncation RPTPα variant and mutational analysis of the D1/D2 interfaces, we show that D2 inhibits RPTPα phosphatase activity and identified a 405PFTP408 motif in D1 that mediates the inhibitory effect of D2. Expression of the gain-of-function F406A/T407A RPTPα variant in HEK293T cells enhanced SRC activation, supporting the relevance of our proposed D2-mediated regulation mechanism in cell signaling. There is emerging interest in the development of allosteric inhibitors of RPTPs but a scarcity of validated allosteric sites for RPTPs. The results of our study not only shed light on the regulatory role of RPTP-D2 domains, but also provide a potentially useful tool for the discovery of chemical probes targeting RPTPα and other RPTPs.

An unexpected 2-histidine phosphoesterase activity of suppressor of T-cell receptor signaling protein 1 contributes to the suppression of cell signaling.

The suppressor of T-cell receptor (TCR) signaling (Sts) proteins Sts-1 and Sts-2 suppress receptor-mediated signaling pathways in various immune cells, including the TCR pathway in T cells and the Dectin-1 signaling pathway in phagocytes. As multidomain enzymes, they contain an N-terminal ubiquitin-association domain, a central Src homology 3 domain, and a C-terminal histidine phosphatase domain. Recently, a 2-histidine (2H) phosphoesterase motif was identified within the N-terminal portion of Sts. The 2H phosphoesterase motif defines an evolutionarily ancient protein domain present in several enzymes that hydrolyze cyclic phosphate bonds on different substrates, including cyclic nucleotides. It is characterized by two invariant histidine residues that play a critical role in catalytic activity. Consistent with its assignment as a phosphoesterase, we demonstrate here that the Sts-1 2H phosphoesterase domain displays catalytic, saturable phosphodiesterase activity toward the dinucleotide 2',3'-cyclic NADP. The enzyme exhibited a high degree of substrate specificity and selectively generated the 3'-nucleotide as the sole product. Sts-1 also had phosphodiesterase catalytic activity toward a 5-mer RNA oligonucleotide containing a 2',3'-cyclic phosphate group at its 3' terminus. To investigate the functional significance of Sts-1 2H phosphoesterase activity, we generated His-to-Ala variants and examined their ability to negatively regulate cellular signaling pathways. Substitution of either conserved histidine compromised the ability of Sts-1 to suppress signaling pathways downstream of both the TCR and the Dectin-1 receptor. Our results identify a heretofore unknown cellular enzyme activity associated with Sts-1 and indicate that this catalytic activity is linked to specific cell-signaling outcomes.

PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.

Phosphatases of regenerating liver (PRLs) are markers of cancer and promote tumor growth. They have been implicated in a variety of biochemical pathways but the physiologically relevant target of phosphatase activity has eluded 20 years of investigation. Here, we show that PRL3 catalytic activity is not required in a mouse model of metastasis. PRL3 binds and inhibits CNNM4, a membrane protein associated with magnesium transport. Analysis of PRL3 mutants specifically defective in either CNNM-binding or phosphatase activity demonstrate that CNNM binding is necessary and sufficient to promote tumor metastasis. As PRLs do have phosphatase activity, they are in fact pseudo-pseudophosphatases. Phosphatase activity leads to formation of phosphocysteine, which blocks CNNM binding and may play a regulatory role. We show levels of PRL cysteine phosphorylation vary in response to culture conditions and in different tissues. Examination of related protein phosphatases shows the stability of phosphocysteine is a unique and evolutionarily conserved property of PRLs. The demonstration that PRL3 functions as a pseudophosphatase has important ramifications for the design of PRL inhibitors for cancer.

Functional interrogation and therapeutic targeting of protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) counteract the enzymatic activity of protein tyrosine kinases to modulate levels of both normal and disease-associated protein tyrosine phosphorylation. Aberrant activity of PTPs has been linked to the progression of many disease states, yet no PTP inhibitors are currently clinically available. PTPs are without a doubt a difficult drug target. Despite this, many selective, potent, and bioavailable PTP inhibitors have been described, suggesting PTPs should once again be looked at as viable therapeutic targets. Herein, we summarize recently discovered PTP inhibitors and their use in the functional interrogation of PTPs in disease states. In addition, an overview of the therapeutic targeting of PTPs is described using SHP2 as a representative target.

A New Paradigm for KIM-PTP Drug Discovery: Identification of Allosteric Sites with Potential for Selective Inhibition Using Virtual Screening and LEI Analysis.

The kinase interaction motif protein tyrosine phosphatases (KIM-PTPs), HePTP, PTPSL and STEP, are involved in the negative regulation of mitogen-activated protein kinase (MAPK) signalling pathways and are important therapeutic targets for a number of diseases. We have used VSpipe, a virtual screening pipeline, to identify a ligand cluster distribution that is unique to this subfamily of PTPs. Several clusters map onto KIM-PTP specific sequence motifs in contrast to the cluster distribution obtained for PTP1B, a classic PTP that mapped to general PTP motifs. Importantly, the ligand clusters coincide with previously reported functional and substrate binding sites in KIM-PTPs. Assessment of the KIM-PTP specific clusters, using ligand efficiency index (LEI) plots generated by the VSpipe, ascertained that the binders in these clusters reside in a more drug-like chemical-biological space than those at the active site. LEI analysis showed differences between clusters across all KIM-PTPs, highlighting a distinct and specific profile for each phosphatase. The most druggable cluster sites are unexplored allosteric functional sites unique to each target. Exploiting these sites may facilitate the delivery of inhibitors with improved drug-like properties, with selectivity amongst the KIM-PTPs and over other classical PTPs.

Substrate Activation of the Low-Molecular Weight Protein Tyrosine Phosphatase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis is known to express a low-molecular weight protein tyrosine phosphatase. This enzyme, denoted as MptpA (Mycobacterium protein tyrosine phosphatase A), is essential for the pathogen to escape the host immune system and therefore represents a target for the search of antituberculosis drugs. MptpA was shown to undergo a conformational transition during catalysis, leading to the closure of the active site, which is by this means poised to the chemical step of dephosphorylation. Here we show that MptpA is subjected to substrate activation, triggered by p-nitrophenyl phosphate or by phosphotyrosine. Moreover, we show that the enzyme is also activated by phosphoserine, with serine being ineffective in enhancing MptpA activity. In addition, we performed assays under pre-steady-state conditions, and we show here that substrate activation is kinetically coupled to the closure of the active site. Surprisingly, when phosphotyrosine was used as a substrate, MptpA did not obey Michealis-Menten kinetics, but we observed a sigmoidal dependence of the reaction velocity on substrate concentration, suggesting the presence of an allosteric activating site in MptpA. This site could represent a promising target for the screening of MptpA inhibitors exerting their action independently of the binding to the enzyme active site.

Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription.

The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II-mediated transcription. During transcriptional elongation, phosphoryl-Ser5 (pSer5) is gradually dephosphorylated by CTD phosphatases, whereas Ser2 phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, here we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer5 at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer2 and pSer5 CTDs. We found that Ssu72 dephosphorylates pSer5 effectively but only has low activities toward pSer7 and pSer2 The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight ß-turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr1 next to Ser2, prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr1 further prohibited Ssu72 binding to pSer2 and thereby prevented untimely Ser2 dephosphorylation. Our results reveal critical roles for Tyr1 in differentiating the phosphorylation states of Ser2/Ser5 of CTD in RNA polymerase II that occur at different stages of transcription.

The BPtpA protein from Burkholderia cenocepacia belongs to a new subclass of low molecular weight protein tyrosine phosphatases.

Low molecular weight protein tyrosine phosphatases (LMW-PTP) are ubiquitous enzymes found across a spectrum of genera from prokaryotes to higher eukaryotes. LMW-PTP belong to the Cys-based PTP class II protein family. Here, we show that LMW-PTP can be categorized into two different groups, referred as class II subdivision I (class II.I) and subdivision II (class II.II). Using BPtpA from the opportunistic pathogen Burkholderia cenocepacia, as a representative member of the LMW-PTP class II.I, we demonstrated that four conserved residues (W47, H48, D80, and F81) are required for enzyme function. Guided by an in silico model of BPtpA, we show that the conserved residues at α3-helix (D80 and F81) contribute to protein stability, while the other conserved residues in the W-loop (W47 and H48) likely play a role in substrate recognition. Overall, our results provide new information on LMW-PTP protein family and establish B. cenocepacia as a suitable model to investigate how substrates are recognized and sorted by these proteins.

Probing biological mechanisms with chemical tools.

Traditionally small molecules have mainly been used to inhibit biochemical activities of proteins, however such compounds can also be used to change the conformational energy landscape of proteins. Tool compounds that modulate protein conformations often reveal unexpected biological mechanisms, which have therapeutic potential. We discuss two examples where screening hits were found to bind to unexpected binding pockets on well known proteins, establishing new routes for the inhibition of proteins that were thought to be undruggable.

Structural Insights into the Active Site Formation of DUSP22 in N-loop-containing Protein Tyrosine Phosphatases.

Cysteine-based protein tyrosine phosphatases (Cys-based PTPs) perform dephosphorylation to regulate signaling pathways in cellular responses. The hydrogen bonding network in their active site plays an important conformational role and supports the phosphatase activity. Nearly half of dual-specificity phosphatases (DUSPs) use three conserved residues, including aspartate in the D-loop, serine in the P-loop, and asparagine in the N-loop, to form the hydrogen bonding network, the D-, P-, N-triloop interaction (DPN-triloop interaction). In this study, DUSP22 is used to investigate the importance of the DPN-triloop interaction in active site formation. Alanine mutations and somatic mutations of the conserved residues, D57, S93, and N128 substantially decrease catalytic efficiency (kcat/KM) by more than 102-fold. Structural studies by NMR and crystallography reveal that each residue can perturb the three loops and induce conformational changes, indicating that the hydrogen bonding network aligns the residues in the correct positions for substrate interaction and catalysis. Studying the DPN-triloop interaction reveals the mechanism maintaining phosphatase activity in N-loop-containing PTPs and provides a foundation for further investigation of active site formation in different members of this protein class.

Molecular Architecture of the Inositol Phosphatase Siw14.

Siw14 is a recently discovered inositol phosphatase implicated in suppressing prion propagation in Saccharomyces cerevisiae. In this paper, we used hybrid structural methods to decipher Siw14 molecular architecture. We found the protein exists in solution as an elongated monomer that is ∼140 Šin length, containing an acidic N-terminal domain and a basic C-terminal dual-specificity phosphatase (DSP) domain, structurally similar to the glycogen phosphatase laforin. The two domains are connected by a protease susceptible linker and do not interact in vitro. The crystal structure of Siw14-DSP reveals a highly basic phosphate-binding loop and an ∼10 Šdeep substrate-binding crevice that evolved to dephosphorylate pyro-phosphate moieties. A pseudoatomic model of the full-length phosphatase generated from solution, crystallographic, biochemical, and modeling data sheds light on the interesting zwitterionic nature of Siw14, which we hypothesized may play a role in discriminating negatively charged inositol phosphates.