From phosphorylase to phosphorylase kinase.
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Animals↗
Biomedical subjects
D Barford
Publications and source records attributed to D Barford.
Small molecule interactions with protein-tyrosine phosphatase PTP1B and their use in inhibitor design.
We have previously shown that a small peptide bearing the hydrolytically stable phosphotyrosyl (pTyr) mimetic, (difluorophosphonomethyl) phenylalanine (F2Pmp), is an extremely potent inhibitor of PTP1B, with an IC50 value of 100 nM [Burke, T. R., Kole, H. K., & Roller, P. P. (1994) Biochem. Biophys. Res. Commun. 204, 129-134]. We further demonstrated that removal of the peptide portion and incorporation of the difluorophosphonomethyl moiety onto a naphthalene ring system, but not a phenyl ring system, resulted in good inhibitory potency [Kole, H. K., Smyth, M. S., Russ, P. L., & Burke, T. R., Jr. (1995) Biochem, J. 311, 1025-1031]. In order to understand the structural basis for this inhibition, and to aid in the design of further analogs, we solved the X-ray structure of [1, 1-difluoro-1-(2-naphthalenyl)-methyl]phosphonic acid (6) complexed within the catalytic site of PTP1B, solved to 2.3 A resolution. In addition to showing the manner in which the phosphonate group is held within the catalytic site, the X-ray structure also revealed extensive hydrophobic interactions with the naphthalene ring system, beyond that possible with an analog bearing a single phenyl ring. It is further evident that, of the two fluorine atoms, the pro-R alpha-fluorine interacts with the enzyme to a significantly greater degree than the pro-S alpha-fluorine, forming a hydrogen bond to Phe 182. On the basis of a computer-assisted molecular modeling analysis, it was determined that addition of a hydroxyl to the naphthyl 4-position, giving [1, 1-difluoro-1-[2-(4-hydroxynaphthalenyl)] methyl]phosphonic acid (8), could potentially replace a water molecule situated in the PTP1B-6 complex, thereby allowing new hydrogen-bonding interactions with Lys 120 and Tyr 46. Compound 8 was therefore prepared and found to exhibit a doubling of affinity (Ki = 94 microM) relative to parent unsubstituted 6 (Ki = 179 microM), supporting, in principle, the development of high-affinity ligands based on molecular modeling analysis of the enzyme-bound parent.
Binding Sites↗
Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.
Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.
Adenylyl Cyclases↗
Further evidence that inhibitor-2 acts like a chaperone to fold PP1 into its native conformation.
The gamma1-isoform of protein phosphatase-1 expressed in Escherichia coli (PP1gamma) and the native PP1 catalytic subunit (PP1C) isolated from skeletal muscle dephosphorylated Ser-14 of glycogen phosphorylase at comparable rates. In contrast, PP1gamma dephosphorylated several tyrosine-phosphorylated proteins at similar rates to authentic protein tyrosine phosphatases (PTPases), but native PP1C was almost inactive towards these substrates. The phosphorylase phosphatase (PhP) and PTPase activities of PP1gamma were inhibited by vanadate with IC50 values (30-100 microM) comparable to authentic PTPases, whereas the PhP activity of native PP1C was insensitive to vanadate. PP1gamma lost its PTPase activity, and its PhP activity became insensitive to vanadate, after interaction with inhibitor-2, followed by the reversible phosphorylation of inhibitor-2 at Thr-72. These findings support and extend the hypothesis that inhibitor-2 functions like a chaperone to fold PP1 into its native conformation, and suggest that the correct folding of PP1 may be critical to prevent the uncontrolled dephosphorylation of cellular phosphotyrosine residues.
Amino Acid Sequence↗
Molecular mechanisms of the protein serine/threonine phosphatases.
The dephosphorylation of proteins on their serine, threonine and tyrosine residues is catalysed by three families of protein phosphatases that regulate numerous intracellular processes. Diversity of structure within a family is generated by targeting and regulatory subunits and domains. Structural studies of these enzymes have revealed that although the two families of protein Ser/Thr phosphatases are unrelated in sequence, the architecture of their catalytic domains is remarkably similar and distinct from the protein tyrosine phosphatases. Insights into the molecular mechanisms of catalysis and regulation of these enzymes have been obtained.
Binding Sites↗
Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate.
Protein phosphatase 1 (PP1) is a serine/threonine protein phosphatase that is essential in regulating diverse cellular processes. Here we report the crystal structure of the catalytic subunit of human PP1 gamma 1 and its complex with tungstate at 2.5 A resolution. The anomalous scattering from tungstate was used in a multiple wavelength anomalous dispersion experiment to derive crystallographic phase information. The protein adopts a single domain with a novel fold, distinct from that of the protein tyrosine phosphatases. A di-nuclear ion centre consisting of Mn2+ and Fe2+ is situated at the catalytic site that binds the phosphate moiety of the substrate. Proton-induced X-ray emission spectroscopy was used to identify the nature of the ions bound to the enzyme. The structural data indicate that dephosphorylation is catalysed in a single step by a metal-activated water molecule. This contrasts with other phosphatases, including protein tyrosine phosphatases, acid and alkaline phosphatases which form phosphoryl-enzyme intermediates. The structure of PP1 provides insight into the molecular mechanism for substrate recognition, enzyme regulation and inhibition of this enzyme by toxins and tumour promoters and a basis for understanding the expanding family of related phosphatases which include PP2A and PP2B (calcineurin).
Amino Acid Sequence↗
Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B.
The crystal structures of a cysteine-215-->serine mutant of protein tyrosine phosphatase 1B complexed with high-affinity peptide substrates corresponding to an autophosphorylation site of the epidermal growth factor receptor were determined. Peptide binding to the protein phosphatase was accompanied by a conformational change of a surface loop that created a phosphotyrosine recognition pocket and induced a catalytically competent form of the enzyme. The phosphotyrosine side chain is buried within the period and anchors the peptide substrate to its binding site. Hydrogen bonds between peptide main-chain atoms and the protein contribute to binding affinity, and specific interactions of acidic residues of the peptide with basic residues on the surface of the enzyme confer sequence specificity.
Binding Sites↗
Protein-tyrosine phosphatase inhibition by a peptide containing the phosphotyrosyl mimetic, L-O-malonyltyrosine.
Peptides containing phosphonate based non-hydrolyzable phosphotyrosyl (pTyr) mimetics previously have been shown to be competitive inhibitors of protein-tyrosine phosphatases (PTPs). These agents suffer from low cellular penetration which is partially attributable to ionization of the phosphonate group at physiological pH. We have developed the non-phosphorus containing pTyr mimetic, L-O-malonyltyrosine (L-OMT) and herein demonstrate using a PTP 1B enzyme assay that it is superior to phosphonomethyl phenylalanine (Pmp) as a pTyr mimetic when incorporated into the hexamer peptide Ac-D-A-D-E-X-L-amide (X = D,L-Pmp, IC50 = 200 microM; X = L-OMT, IC50 = 10 microM). Prodrug protection of L-OMT as its carboxylic acid diester could potentially increase cellular penetration, thereby making this a valuable reagent for cellular studies.
Amino Acid Sequence↗
Protein phosphatases.
Protein phosphatases are signal transducing enzymes that dephosphorylate cellular phosphoproteins. The recently determined crystal structures of protein tyrosine and serine/threonine phosphatases reveal that these proteins adopt distinct structures and catalyze dephosphorylation reactions by means of different enzymatic mechanisms. Insights into the basis for substrate specificity and enzyme regulation can also be gained from these crystal structures.
Humans↗
Protein tyrosine phosphatases take off.
Protein tyrosine phosphatases (PTPs) are a family of signal transduction enzymes that dephosphorylate phosphotyrosine containing proteins. Structural and kinetic studies provide a molecular understanding of how these enzymes regulate a wide range of intracellular processes.
Amino Acid Sequence↗
Purification and crystallization of the catalytic domain of human protein tyrosine phosphatase 1B expressed in Escherichia coli.
The amino-terminal 321 residues encoding the catalytic domain of human protein tyrosine phosphatase 1B (molecular mass 37 kDa) has been expressed in Escherichia coli, purified to homogeneity and crystallized. The crystals diffract to 2.4 A resolution when exposed to synchrotron radiation and belong to space group P3(1)21 (or its enantiomorph P3(2)21) with a = 88.4 A, b = 88.4 A, c = 104.0 A, alpha = beta = 90.0 degrees, gamma = 120.0 degrees. There is one molecule of protein tyrosine phosphatase 1B per asymmetric unit and the crystal form is suitable for the determination of the atomic structure of the enzyme.
Amino Acid Sequence↗
Crystal structure of human protein tyrosine phosphatase 1B.
Protein tyrosine phosphatases (PTPs) constitute a family of receptor-like and cytoplasmic signal transducing enzymes that catalyze the dephosphorylation of phosphotyrosine residues and are characterized by homologous catalytic domains. The crystal structure of a representative member of this family, the 37-kilodalton form (residues 1 to 321) of PTP1B, has been determined at 2.8 A resolution. The enzyme consists of a single domain with the catalytic site located at the base of a shallow cleft. The phosphate recognition site is created from a loop that is located at the amino-terminus of an alpha helix. This site is formed from an 11-residue sequence motif that is diagnostic of PTPs and the dual specificity phosphatases, and that contains the catalytically essential cysteine and arginine residues. The position of the invariant cysteine residue within the phosphate binding site is consistent with its role as a nucleophile in the catalytic reaction. The structure of PTP1B should serve as a model for other members of the PTP family and as a framework for understanding the mechanism of tyrosine dephosphorylation.
Amino Acid Sequence↗
Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases.
A multiple sequence alignment of 44 serine/threonine-specific protein phosphatases has been performed. This reveals the position of a common conserved catalytic core, the location of invariant residues, insertions and deletions. The multiple alignment has been used to guide and improve a consensus secondary-structure prediction for the common catalytic core. The location of insertions and deletions has aided in defining the positions of surface loops and turns. The prediction suggests that the core protein phosphatase structure comprises two domains: the first has a single, beta sheet flanked by alpha helices, while the second is predominantly alpha helical. Knowledge of the core secondary structures provides a guide for the design of site-directed-mutagenesis experiments that will not disrupt the native phosphatase fold. A sequence similarity between eukaryotic serine/threonine protein phosphatases and the Escherichia coli diadenosine tetraphosphatase has been identified. This extends over the N-terminal 100 residues of bacteriophage phosphatases and E. coli diadenosine tetraphosphatase. Residues which are invariant amongst these classes are likely to be important in catalysis and protein folding. These include Arg92, Asn138, Asp59, Asp88, Gly58, Gly62, Gly87, Gly93, Gly137, His61, His139 and Val90 and fall into three clusters with the consensus sequences GD(IVTL)HG, GD(LYF)V(DA)RG and GNH, where brackets surround alternative amino acids. The first two consensus sequences are predicted to fall in the beta-alpha and beta-beta loops of a beta-alpha-beta-beta secondary-structure motif. This places the predicted phosphate-binding site at the N-terminus of the alpha helix, where phosphate binding may be stabilised by the alpha-helix dipole.
Acid Anhydride Hydrolases↗
Co-crystallization of the catalytic subunit of the serine/threonine specific protein phosphatase 1 from human in complex with microcystin LR.
The catalytic subunit of the serine/threonine specific protein phosphatase 1 from human (molecular mass 37 KDa) has been co-crystallized in complex with the cyanobacterial toxin microcystin LR (molecular mass 1 kDa). The crystals diffract to a resolution of 2.8 A when exposed to synchrotron radiation and belong to space group P2(1)2(1)2 with a = 109.5 A, b = 90.6 A, c = 38.7 A. There is one molecule of protein phosphatase 1 per asymmetric unit. The crystal form is suitable for the determination of the atomic structure of protein phosphatase 1.
Crystallization↗
Electrostatic effects in the control of glycogen phosphorylase by phosphorylation.
Electrostatic effects are important in the initial activation mechanism of glycogen phosphorylase by phosphorylation. Analysis of the electrostatic surface potential of glycogen phosphorylase with the program GRASP shows that in the unphosphorylated state, the N-terminal 20 residues, which include a number of basic amino acids, are located close to a position on the surface of the molecule that is highly acidic. Upon phosphorylation by phosphorylase kinase at Ser 14, the N-terminal residues change their position and conformation so that the Ser-P is directed away from the acidic patch and to an intersubunit site where 2 arginines bind the phosphate. This recognition site is created through tertiary and quaternary structural changes that accompany the activation mechanism.
Computer Simulation↗
Expression, purification, crystallization, and biochemical characterization of a recombinant protein phosphatase.
A protein phosphatase (PPase) from the bacteriophage lambda was overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity yielding approximately 17 mg of enzyme from a single liter of bacterial culture. Biochemical characterization of the enzyme showed that it required Mn2+ or Ni2+ as an activator. The recombinant enzyme was active toward serine, threonine, and tyrosine phosphoproteins and phosphopeptides. Surprisingly, the bacterial histidyl phosphoprotein, NRII, was also dephosphorylated by the lambda-PPase. The lambda-PPase shares a number of kinetic and structural properties with the eukaryotic Ser/Thr phosphatases, suggesting that the lambda-PPase will serve as a good model for structure-function studies. Crystallization of the recombinant purified lambda-PPase yielded monoclinic crystals. The crystals diffract to 4.0 A when exposed to synchrotron x-ray radiation.
Amino Acid Sequence↗
Crystallographic binding studies on the allosteric inhibitor glucose-6-phosphate to T state glycogen phosphorylase b.
Glucose-6-phosphate is an important allosteric inhibitor of glycogen phosphorylase b that restrains the enzyme in the inactive state in resting muscle. A crystallographic binding study by diffusion of glucose-6-phosphate into performed crystals of T state phosphorylase b has been carried out at 2.3 A resolution and the structure refined by restrained crystallographic least-squares and simulated annealing to give a crystallographic R-value of 0.203. The inhibitor binds at the AMP allosteric effector site at the subunit-subunit interface of the dimer. The phosphate groups of the glucose-6-phosphate and AMP occupy partially overlapping sites and make similar contacts to two arginine residues (Arg309 and Arg310) but in glucose-6-phosphate there is a contact to a third arginine (Arg242). The glucopyranose of glucose-6-phosphate and the adenine ribose of AMP occupy different positions. Including the contacts to the three arginine residues by the phosphate group, the glucose-6-phosphate makes a total of 11 hydrogen-bonds to the enzyme and all but one of these are to charged groups. The O-2 hydroxyl hydrogen-bonds to the main-chain carbonyl oxygen of Val40' from the other subunit and this interaction appears important for the allosteric response. There are substantial conformational changes both in the vicinity of the glucose-6-phosphate (involving for example Phe196 and Arg309) and at the subunit interface (involving residues 42' to 51' and 192 to 196). These shifts tighten the binding of the inhibitor and the interface. Comparison of the glucose-6-phosphate complex with the T state native phosphorylase b and the R state phosphorylase a structures shows that there is a graded response from T state glucose-6-phosphate complex through T state phosphorylase b to R state phosphorylase a that suggests that glucose-6-phosphate promotes a tight structure that is more "tensed" than native T state phosphorylase b. The results show how the same allosteric effector site can exhibit a tight binding site for the activator AMP in the R state structure and a tight binding site for glucose-6-phosphate in the modified T state structure.
Allosteric Regulation↗
Solid state 31P cross-polarization/magic angle sample spinning nuclear magnetic resonance of crystalline glycogen phosphorylase b.
(31)P cross-polarization/magic angle sample spinning nuclear magnetic resonance spectra have been obtained for pyridoxal 5'-phosphate (PLP) bound to glycogen phosphorylase b (GPb) in two different crystalline forms, monoclinic and tetragonal. Analysis of the intensities of the spinning sidebands in the nuclear magnetic resonance spectra has enabled estimates of the principal values of the (31)P chemical shift tensors to be obtained. Differences between the two sets of values suggest differences in the environment of the phosphate moiety of the pyridoxal phosphate in the two crystalline forms. The tensor for the tetragonal crystalline form, T state GPb, is fully consistent with those found for dianionic phosphate groups in model compounds. The spectrum for the monoclinic crystalline form, R state GPb, although closer to that of dianionic than monoanionic model phosphate compounds, deviates significantly from that expected for a simple dianion or monoanion. This is likely to result from specific interactions between the PLP phosphate group and residues in its binding site in the protein. A possible explanation for the spectrum of the monoclinic crystals is that the shift tensor is averaged by a proton exchange process between different ionization states of the PLP associated with the presence of a sulfate ion bound in the vicinity of the PLP.
Allosteric Site↗