Jeanne A. Stuckey

Form and Function in Protein Dephosphorylation

In contrast to the catalytic mechanism employed by PPs, the PTPs proceed through a phosphoenzyme intermediate. The enzymatic reaction involves phosphoryl-cysteine intermediate formation after nucleophilic attack of the phosphorus atom of the substrate by the thiolate anion of cysteine (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996). The reaction can be represented as a two-step chemical process: phosphoryl transfer to the enzyme accompanied by rapid release of dephosphorylated product (denoted by rate constant k(formation) in Equation 1); and hydrolysis of the thiol-phosphate intermediate concomitant with rapid release of phosphate (denoted by rate constant k(hydrolysis) in Equation 1). To form the catalytically competent complex ES, the enzyme binds and reacts with the dianion of phosphate–containing substrate (Figure 3AFigure 3A). On the enzyme an aspartic acid must be protonated and the nucleophilic cysteine must be unprotonated (thiolate anion) for phosphoryl transfer to the enzyme (3xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all References, 20xZhang, Z.-Y. J. Biol. Chem. 1995; 270: 11199–11204Crossref | PubMed | Scopus (86)See all References). In the Michaelis complex (Jia et al. 1995xJia, Z, Barford, D, Flint, A.J, and Tonks, N.K. Science. 1995; 268: 1754–1758Crossref | PubMedSee all ReferencesJia et al. 1995), the three nonbridging oxygens of the phosphoryl group are coordinated by bidentate hydrogen bonds to the guanidinium group of arginine and by the backbone amide N-H groups of the active-site loop (Figure 3AFigure 3A) . Situated directly underneath the phosphoryl group and at the base of the active-site cleft, is the nucleophilic cysteine thiolate anion (shown in green, Figure 3AFigure 3A). The substrate (shown in yellow, Figure 3AFigure 3A) is positioned such that attack of the thiolate is directly in line with the P-O bond and ideally situated for efficient expulsion of the leaving group. To further enhance leaving group expulsion, near the top of the cleft the aspartic acid is positioned to act as a general acid by protonating the leaving group phenolic oxygen. The aspartic acid is found on a separate loop (shown in yellow, Figure 2Figure 2) that was shown to be flexible for both Yersinia PTP (Stuckey et al. 1994xStuckey, J.A, Schubert, H.L, Fauman, E.B, Zhang, Z.-Y, Dixon, J.E, and Saper, M.A. Nature. 1994; 370: 571–575Crossref | PubMed | Scopus (303)See all ReferencesStuckey et al. 1994) and PTP1B (Jia et al. 1995xJia, Z, Barford, D, Flint, A.J, and Tonks, N.K. Science. 1995; 268: 1754–1758Crossref | PubMedSee all ReferencesJia et al. 1995). When the enzyme is complexed with phosphorylated substrates, this loop folds over the active site, bringing the aspartic acid into position for leaving group protonation. In the open conformation, the loop is flipped away from the active site and the aspartic acid is approximately 8–12 A removed from its location in the Michaelis complex (Stuckey et al. 1994xStuckey, J.A, Schubert, H.L, Fauman, E.B, Zhang, Z.-Y, Dixon, J.E, and Saper, M.A. Nature. 1994; 370: 571–575Crossref | PubMed | Scopus (303)See all ReferencesStuckey et al. 1994). It is not yet known whether the analogous loop in VHR is capable of such dramatic movement. In the sulfate-bound complex of VHR, the loop does not cover the active site to the same extent as in the other two PTP crystal structures (Yuvaniyama, et al. 1996xYuvaniyama, J, Denu, J.M, Dixon, J.E, and Saper, M.A. Science. 1996; 272: 1328–1331Crossref | PubMedSee all ReferencesYuvaniyama, et al. 1996).Figure 3Catalytic Mechanism of Protein Tyrosine Phosphatases: Enzyme–Substrate Complex and Phosphoenzyme Intermediate(A) A model of the enzyme-substrate complex of PTP derived from two X-ray crystallographic models: Cys-ser mutant of PTP1B complexed with phosphotyrosine (Jia et al. 1995xJia, Z, Barford, D, Flint, A.J, and Tonks, N.K. Science. 1995; 268: 1754–1758Crossref | PubMedSee all ReferencesJia et al. 1995), and Yersinia PTP complexed with vanadate (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996). The backbone atoms of the active site loop from cysteine to arginine are shown as a ball-and-stick model. The phospho-tyrosine substrate (shown in yellow) is bound to the center of the active-site motif with the protonated general acid (Asp) lying within hydrogen bonding distance of the scissile oxygen of the substrate. The dianion of the phosphoryl group is coordinated by the nitrogens of the arginine side chain and by the amide groups of the active site motif. The thiolate anion of cysteine is poised for nucleophilic attack. Individuals atoms are represented as spheres with the following colors: Cα and carbonyl carbons, gray; oxygens, red; nitrogens, blue; phosphorus, magenta; sulfur, green; and hydrogens, orange (not found in original crystal structures). Dashed lines represent hydrogen bonds.(B) A model of the phosphoenzyme intermediate of PTP derived in a similar manner as (A) with the same atom colors as (A). The phosphorus is covalently bound to the Sγ of the cysteine. A water molecule (Wat) is in a position such that aspartic acid can abstract the proton. Figures were drawn using Molscript and Raster3D.View Large Image | View Hi-Res Image | Download PowerPoint SlideTo explore the transition-state structure for catalysis, heavy atom kinetic isotope effects have been measured with PTP1, Yersinia PTP and VHR (Hengge et al. 1996xHengge, A.C, Denu, J.M, and Dixon, J.E. Biochemistry. 1996; 35: 7084–7092Crossref | PubMed | Scopus (61)See all ReferencesHengge et al. 1996). The kinetic isotope effects indicate that P-O bond cleavage is far advanced and there is little bond formation to the nucleophile cysteine during the transition state. It was also demonstrated that proton transfer from aspartic acid to the bridging oxygen is concomitant with P-O cleavage, such that no charge is developed on the leaving group. Consistent with concomitant proton transfer, leaving group pKa values have little effect on the rate of phosphoryl transfer (20xZhang, Z.-Y. J. Biol. Chem. 1995; 270: 11199–11204Crossref | PubMed | Scopus (86)See all References, 3xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all References). When aspartic acid is replaced by asparagine, phosphopeptides and phosphotyrosine are 100-fold worse than substrates with good leaving groups such as p-nitrophenylphosphate, a commonly employed artificial substrate. Thus, with physiological substrates, proton donation by aspartic acid is more critical for efficient catalysis than with labile artificial substrates.Once the phosphoenzyme intermediate is formed, it must undergo hydrolysis by water to result in the turnover of the enzyme (Figure 3Figure 3). Activation of the water molecule by a general base would be expected to facilitate hydrolysis. Mutagenic studies with VHR have shown that the conserved aspartic acid enhances intermediate hydrolysis (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996), suggesting that the aspartic acid may function as the general base by proton abstraction from a water molecule. Further support for this idea comes from the X-ray structure of Yersinia PTP solved with the inhibitor vanadate covalently bound to cysteine (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996). With trigonal bipyramidal geometry, the vanadate mimics the transition for both chemical steps and underscores the importance of conserved arginine and aspartic acid residues. Similar to the ES complex, two of the equatorial oxygens of vanadate are hydrogen bonded to arginine in a bidendate fashion. The aspartic acid makes a hydrogen bond of 2.8 A to the apical oxygen of vanadate, consistent with the role as general acid/base.The recently solved structures of protein phosphatases have greatly advanced our knowledge of the molecular mechanism of catalysis and substrate specificity. Understanding the molecular events of these essential reactions will lead to a better understanding of the specific physiological functions of the protein phosphatases. Also, a detailed description of the inherent differences between and among PTPs and PPs will undoubtedly augment the search for and design of specific phosphatase inhibitors.Because of reference limitations imposed in minireviews, we wish to acknowledge the important contributions made by other investigators whose work we were unable to cite. Reference to this work can be found within the cited publications of many of the selected reading articles.

Interaction between Pyrin and the Apoptotic Speck Protein (ASC) Modulates ASC-induced Apoptosis

Patients with familial Mediterranean fever suffer sporadic inflammatory attacks characterized by fever and intense pain (in joints, abdomen, or chest). Pyrin, the product of theMEFV locus, is a cytosolic protein whose function is unknown. Using pyrin as a “bait” to probe a yeast two-hybrid library made from neutrophil cDNA, we isolatedapoptotic speck protein containing a caspase recruitment domain (CARD) (ASC), a proapoptotic protein that induces the formation of large cytosolic “specks” in transfected cells. We found that when HeLa cells are transfected with ASC, specks are formed. After co-transfection of cells with ASC plus wild type pyrin, an increase in speck-positive cells is found, and speck-positive cells show increased survival. Immunofluorescence studies show that pyrin co-localizes with ASC in specks. Speck localization requires exon 1 of pyrin, but exon 1 alone of pyrin does not result in an increase in the number of specks. Exon 1 of pyrin and exon 1 of ASC show 42% sequence similarity and resemble death domain-related structures in modeling studies. These findings link pyrin to apoptosis pathways and suggest that the modulation of cell survival may be a component of the pathophysiology of familial Mediterranean fever.

Crystal structure of a phospholipase D family member

The first crystal structure of a phospholipase D (PLD) family member has been determined at 2.0 Å resolution. The PLD superfamily is defined by a common sequence motif, HxK(x)4D(x)6GSxN, and includes enzymes involved in signal transduction, lipid biosynthesis, endonucleases and open reading frames in pathogenic viruses and bacteria. The crystal structure suggests that residues from two sequence motifs form a single active site. A histidine residue from one motif acts as a nucleophile in the catalytic mechanism, forming a phosphoenzyme intermediate, whereas a histidine residue from the other motif appears to function as a general acid in the cleavage of the phosphodiester bond. The structure suggests that the conserved lysine residues are involved in phosphate binding. Large-scale genomic sequencing revealed that there are many PLD family members. Our results suggest that all of these proteins may possess a common structure and catalytic mechanism.

Hydrolytic catalysis and structural stabilization in a designed metalloprotein

Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions – a Zn(II) ion which is important for catalytic activity and a Hg(II) ion which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate hydrolysis (pNPA) to within ~100-fold of the efficiency of human carbonic anhydrase (CA)II and is at least 550-fold better than comparable synthetic complexes. Similarly, CO2 hydration occurs with an efficiency within ~500-fold of CAII. While histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO2 hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme uncovers necessary design features for future metalloenzymes containing one or more metals.

SM-164: a novel, bivalent Smac mimetic that induces apoptosis and tumor regression by concurrent removal of the blockade of cIAP-1/2 and XIAP.

Small-molecule Smac mimetics are being developed as a novel class of anticancer drugs. Recent studies have shown that Smac mimetics target cellular inhibitor of apoptosis protein (cIAP)-1/2 for degradation and induce tumor necrosis factor-alpha (TNFalpha)-dependent apoptosis in tumor cells. In this study, we have investigated the mechanism of action and therapeutic potential of two different types of novel Smac mimetics, monovalent SM-122 and bivalent SM-164. Our data showed that removal of cIAP-1/2 by Smac mimetics or small interfering RNA is not sufficient for robust TNFalpha-dependent apoptosis induction, and X-linked inhibitor of apoptosis protein (XIAP) plays a critical role in inhibiting apoptosis induction. Although SM-164 is modestly more effective than SM-122 in induction of cIAP-1/2 degradation, SM-164 is 1,000 times more potent than SM-122 as an inducer of apoptosis in tumor cells, which is attributed to its much higher potency in binding to and antagonizing XIAP. SM-164 induces rapid cIAP-1 degradation and strong apoptosis in the MDA-MB-231 xenograft tumor tissues and achieves tumor regression, but has no toxicity in normal mouse tissues. Our study provides further insights into the mechanism of action for Smac mimetics and regulation of apoptosis by inhibitor of apoptosis proteins. Furthermore, our data provide evidence that SM-164 is a promising new anticancer drug for further evaluation and development.

Crystal Structure of a Phosphoinositide Phosphatase, MTMR2: Insights into Myotubular Myopathy and Charcot-Marie-Tooth Syndrome

Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in approximately 180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.

Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member.

A phospholipase D (PLD) superfamily was recently identified that contains proteins of highly diverse functions with the conserved motif HXKX 4DX 6G(G/S). The superfamily includes a bacterial nuclease, human and plant PLD enzymes, cardiolipin synthases, phosphatidylserine synthases, and the murine toxin from Yersinia pestis (Ymt). Ymt is particularly effective as a prototype for family members containing two conserved motifs, because it is smaller than many other two-domain superfamily enzymes, and it can be overexpressed. Large quantities of pure recombinant Ymt allowed the formation of diffraction-quality crystals for x-ray structure determination. Dimeric Ymt was shown to have PLD-like activity as demonstrated by the hydrolysis of phosphatidylcholine. Ymt also used bis(para-nitrophenol) phosphate as a substrate. Using these substrates, the amino acids essential for Ymt function were determined. Specifically, substitution of histidine or lysine in the conserved motifs reduced the turnover rate of bis(para-nitrophenol) phosphate by a factor of 104 and phospholipid turnover to an undetectable level. The role of the conserved residues in catalysis was further defined by the isolation of a radiolabeled phosphoenzyme intermediate, which identified a conserved histidine residue as the nucleophile in the catalytic reaction. Based on these data, a unifying two-step catalytic mechanism is proposed for this diverse family of enzymes.

SAR405838: An Optimized Inhibitor of MDM2–p53 Interaction That Induces Complete and Durable Tumor Regression

Blocking the oncoprotein murine double minute 2 (MDM2)-p53 protein-protein interaction has long been considered to offer a broad cancer therapeutic strategy, despite the potential risks of selecting tumors harboring p53 mutations that escape MDM2 control. In this study, we report a novel small-molecule inhibitor of the MDM2-p53 interaction, SAR405838 (MI-77301), that has been advanced into phase I clinical trials. SAR405838 binds to MDM2 with K(i) = 0.88 nmol/L and has high specificity over other proteins. A cocrystal structure of the SAR405838:MDM2 complex shows that, in addition to mimicking three key p53 amino acid residues, the inhibitor captures additional interactions not observed in the p53-MDM2 complex and induces refolding of the short, unstructured MDM2 N-terminal region to achieve its high affinity. SAR405838 effectively activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell-cycle arrest and/or apoptosis. At well-tolerated dose schedules, SAR405838 achieves either durable tumor regression or complete tumor growth inhibition in mouse xenograft models of SJSA-1 osteosarcoma, RS4;11 acute leukemia, LNCaP prostate cancer, and HCT-116 colon cancer. Remarkably, a single oral dose of SAR405838 is sufficient to achieve complete tumor regression in the SJSA-1 model. Mechanistically, robust transcriptional upregulation of PUMA induced by SAR405838 results in strong apoptosis in tumor tissue, leading to complete tumor regression. Our findings provide a preclinical basis upon which to evaluate SAR405838 as a therapeutic agent in patients whose tumors retain wild-type p53.

CSAR Benchmark Exercise 2011–2012: Evaluation of Results from Docking and Relative Ranking of Blinded Congeneric Series

The Community Structure–Activity Resource (CSAR) recently held its first blinded exercise based on data provided by Abbott, Vertex, and colleagues at the University of Michigan, Ann Arbor. A total of 20 research groups submitted results for the benchmark exercise where the goal was to compare different improvements for pose prediction, enrichment, and relative ranking of congeneric series of compounds. The exercise was built around blinded high-quality experimental data from four protein targets: LpxC, Urokinase, Chk1, and Erk2. Pose prediction proved to be the most straightforward task, and most methods were able to successfully reproduce binding poses when the crystal structure employed was co-crystallized with a ligand from the same chemical series. Multiple evaluation metrics were examined, and we found that RMSD and native contact metrics together provide a robust evaluation of the predicted poses. It was notable that most scoring functions underpredicted contacts between the hetero atoms (i.e., N, O, S, etc.) of the protein and ligand. Relative ranking was found to be the most difficult area for the methods, but many of the scoring functions were able to properly identify Urokinase actives from the inactives in the series. Lastly, we found that minimizing the protein and correcting histidine tautomeric states positively trended with low RMSD for pose prediction but minimizing the ligand negatively trended. Pregenerated ligand conformations performed better than those that were generated on the fly. Optimizing docking parameters and pretraining with the native ligand had a positive effect on the docking performance as did using restraints, substructure fitting, and shape fitting. Lastly, for both sampling and ranking scoring functions, the use of the empirical scoring function appeared to trend positively with the RMSD. Here, by combining the results of many methods, we hope to provide a statistically relevant evaluation and elucidate specific shortcomings of docking methodology for the community.

The X-ray Crystal Structures of Yersinia Tyrosine Phosphatase with Bound Tungstate and Nitrate MECHANISTIC IMPLICATIONS

X-ray crystal structures of the Yersinia tyrosine phosphatase (PTPase) in complex with tungstate and nitrate have been solved to 2.4-Å resolution. Tetrahedral tungstate, WO2−4, is a competitive inhibitor of the enzyme and is isosteric with the substrate and product of the catalyzed reaction. Planar nitrate, NO−3, is isosteric with the PO3 moiety of a phosphotransfer transition state. The crystal structures of the Yersinia PTPase with and without ligands, together with biochemical data, permit modeling of key steps along the reaction pathway. These energy-minimized models are consistent with a general acid-catalyzed, in-line displacement of the phosphate moiety to Cys403 on the enzyme, followed by attack by a nucleophilic water molecule to release orthophosphate. This nucleophilic water molecule is identified in the crystal structure of the nitrate complex. The active site structure of the PTPase is compared to alkaline phosphatase, which employs a similar phosphomonoester hydrolysis mechanism. Both enzymes must stabilize charges at the nucleophile, the PO3 moiety of the transition state, and the leaving group. Both an associative (bond formation preceding bond cleavage) and a dissociative (bond cleavage preceding bond formation) mechanism were modeled, but a dissociative-like mechanism is favored for steric and chemical reasons. Since nearly all of the 47 invariant or highly conserved residues of the PTPase domain are clustered at the active site, we suggest that the mechanism postulated for the Yersinia enzyme is applicable to all the PTPases.