ABSTRACT
The structure of the Ascaris suum mitochondrial NAD-malic enzyme in binary complex with NAD has been solved to a resolution of 2.3 A by X-ray crystallography. The structure resembles that of the human mitochondrial enzyme determined in complex with NAD [Xu, Y., Bhargava, G., Wu, H., Loeber, G., and Tong, L. (1999) Structure 7, 877-889]. The enzyme is a tetramer comprised of subunits possessing four domains organized in an "open" structure typical of the NAD-bound form. The subunit organization, as in the human enzyme, is a dimer of dimers. The Ascaris enzyme contains 30 additional residues at its amino terminus relative to the human enzyme. These residues significantly increase the interactions that promote tetramer formation and give rise to different subunit-subunit interactions. Unlike the mammalian enzyme, the Ascaris malic enzyme is not regulated by ATP, and no ATP binding site is observed in this structure. Although the active sites of the two enzymes are similar, residues interacting with NAD differ between the two. The structure is discussed in terms of the mechanism and particularly with respect to previously obtained kinetic and site-directed mutagenesis experiments.
Subject(s)
Ascaris/enzymology , Malate Dehydrogenase/chemistry , Mitochondria/enzymology , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray/methods , Humans , Models, Molecular , Molecular Sequence DataABSTRACT
Serpins are irreversible covalent 'suicide' protease inhibitors. In the past two years, important advances in the structural biology of serpins have been forthcoming with the crystal structures of a covalent complex between trypsin and alpha1-antitrypsin, and of a Michaelis encounter complex between trypsin S195A and serpin 1B from Manduca sexta. These structures have helped elucidate many aspects of the mechanism of action of serpins. Also, the crystal structure of the cysteine protease caspase-8 in complex with the inhibitor p35 has revealed a new family of suicide protease inhibitors.
Subject(s)
Serine Proteinase Inhibitors/chemistry , Serpins/chemistry , Caspase 8 , Caspase 9 , Caspases/chemistry , Caspases/metabolism , Protein Conformation , Trypsin , Trypsin Inhibitors/chemistry , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
Serine protease inhibitors (serpins) regulate the activities of circulating proteases. Serpins inhibit proteases by acylating the serine hydroxyl at their active sites. Before deacylation and complete proteolysis of the serpin can occur, massive conformational changes are triggered in the serpin while maintaining the covalent linkage between the protease and serpin. Here we report the structure of a serpin-trypsin Michaelis complex, which we visualized by using the S195A trypsin mutant to prevent covalent complex formation. This encounter complex reveals a more extensive interaction surface than that present in small inhibitor-protease complexes and is a template for modeling other serpin-protease pairs. Mutations of several serpin residues at the interface reduced the inhibitory activity of the serpin. The serine residue C-terminal to the scissile peptide bond is found in a closer than usual interaction with His 57 at the active site of trypsin.
Subject(s)
Manduca/chemistry , Serpins/chemistry , Serpins/metabolism , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/metabolism , Trypsin/chemistry , Trypsin/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , Crystallography, X-Ray , Hydrogen Bonding , Manduca/genetics , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Protein Conformation , Protein Interaction Mapping , Rats , Sequence Alignment , Serpins/genetics , Trypsin/genetics , Trypsin Inhibitors/geneticsABSTRACT
Changes in protein mobility accompany changes in conformation during the trans-activation of enzymes; however, few studies exist that validate or characterize this behavior. In this study, amide hydrogen/deuterium exchange/mass spectrometry was used to probe the conformational flexibility of extracellular signal-regulated protein kinase-2 before and after activation by phosphorylation. The exchange data indicated that extracellular regulated protein kinase-2 activation caused altered backbone flexibility in addition to the conformational changes previously established by x-ray crystallography. The changes in flexibility occurred in regions involved in substrate binding and turnover, suggesting their importance in enzyme regulation.
Subject(s)
Mitogen-Activated Protein Kinase 1/chemistry , Mitogen-Activated Protein Kinase 1/metabolism , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Hydrogen Bonding , Ligands , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phosphorylation , Protein Conformation , Protein Structure, Secondary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Ornithine decarboxylase (ODC) catalyzes the first committed step in the biosynthesis of polyamines, and it has been identified as a drug target for the treatment of African sleeping sickness, caused by Trypanosoma brucei. ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme and an obligate homodimer. X-ray structural analysis of the complex of the T. brucei wild-type enzyme with the product putrescine reveals two structural changes that occur upon ligand binding: Lys-69 is displaced by putrescine and forms new interactions with Glu-94 and Asp-88, and the side chain of Cys-360 rotates into the active site to within 3.4 A of the imine bond. Mutation of Cys-360 to Ala or Ser reduces the k(cat) of the decarboxylation reaction by 50- and 1000-fold, respectively. However, HPLC analysis of the products demonstrates that the mutant enzymes almost exclusively catalyze a decarboxylation-dependent transamination reaction to form pyridoxamine 5-phosphate (PMP) and gamma-aminobutyraldehyde, instead of PLP and putrescine. This side reaction arises when the decarboxylated substrate intermediate is protonated at C4' of PLP instead of at the C(alpha) of substrate. For the reaction catalyzed by the wild-type enzyme, this side reaction occurs infrequently (<0.01% of the turnovers). Single turnover analysis and multiwavelength stopped-flow spectroscopic studies suggest that for the mutant ODCs protonation at C4' occurs either very rapidly or in a concerted reaction with decarboxylation and that the rate-limiting step in the steady-state reaction is Schiff base hydrolysis/product release. These studies demonstrate a role for Cys-360 in the control of the C(alpha) protonation step that catalyzes the formation of the physiological product putrescine. The results further provide insight into the mechanism by which this class of PLP-dependent enzymes controls reaction specificity.
Subject(s)
Mutagenesis, Site-Directed , Ornithine Decarboxylase/chemistry , Ornithine Decarboxylase/genetics , Alanine/genetics , Animals , Binding Sites/genetics , Catalysis , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Cysteine/genetics , Decarboxylation , Kinetics , Models, Molecular , Ornithine Decarboxylase/metabolism , Putrescine/chemistry , Serine/genetics , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Substrate Specificity/genetics , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/geneticsABSTRACT
We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases.
Subject(s)
Lysine/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Catalytic Domain , DNA, Complementary , Minor Histocompatibility Antigens , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Rats , Sequence Homology, Amino Acid , WNK Lysine-Deficient Protein Kinase 1ABSTRACT
The stimulus-dependent nuclear localization of the extracellular-signal- regulated kinases ERK1 and ERK2 is required for many of their actions, including induction of neurites in PC12 cells and transformation of fibroblasts. Phosphorylation of ERK2 causes it to form dimers, and the most flexible portions of the ERK2 molecule provide the surfaces for dimerization. It is thought that dimerization promotes nuclear localization of ERK2 by its effects on import, export or retention in cytoplasmic and nuclear compartments. Dimerization might also influence substrate interactions.
Subject(s)
Mitogen-Activated Protein Kinases/chemistry , Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Nucleus/metabolism , Dimerization , Humans , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1/chemistry , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Phosphorylation , Protein Conformation , Signal TransductionABSTRACT
Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by Trypanosoma brucei. One of the currently used drugs, alpha-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO has been determined by X-ray crystallography to 2.0 A resolution. The protein crystallizes in the space group P2(1) (a = 66.8 A, b = 154.5 A, c = 77.1 A, beta = 90.58 degrees ), with two dimers per asymmetric unit. The initial phasing was done by molecular replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also determined to 2.9 A resolution by molecular replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC is a beta/alpha-barrel, and the C-terminal domain of ODC is a modified Greek key beta-barrel. In comparison to structurally related alanine racemase, the two domains are rotated 27 degrees relative to each other. In addition, two of the beta-strands in the C-terminal domain have exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface are formed primarily by the C-terminal domains, which interact through six aromatic rings that form stacking interactions across the domain boundary. The PLP binding site is formed by the C-termini of beta-strands and loops in the beta/alpha-barrel. In the native structure Lys69 forms a Schiff base with PLP. In both structures, the phosphate of PLP is bound between the seventh and eighth strands forming interactions with Arg277 and a Gly loop (residues 235-237). The pyridine nitrogen of PLP interacts with Glu274. DFMO forms a Schiff base with PLP and is covalently attached to Cys360. It is bound at the dimer interface and the delta-carbon amino group of DFMO is positioned between Asp361 of one subunit and Asp332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 has rotated 145 degrees toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes are observed upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.
Subject(s)
Eflornithine/chemistry , Enzyme Inhibitors/chemistry , Ornithine Decarboxylase Inhibitors , Ornithine Decarboxylase/chemistry , Trypanosoma brucei brucei/enzymology , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Dimerization , Eflornithine/metabolism , Mice , Molecular Sequence Data , Ornithine Decarboxylase/genetics , Ornithine Decarboxylase/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Pyridoxal Phosphate/metabolism , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Substrate SpecificityABSTRACT
BACKGROUND: The reactive center loops (RCL) of serpins undergo large conformational changes triggered by the interaction with their target protease. Available crystallographic data suggest that the serpin RCL is polymorphic, but the relevance of the observed conformations to the competent active structure and the conformational changes that occur on binding target protease has remained obscure. New high-resolution data on an active serpin, serpin 1K from the moth hornworm Manduca sexta, provide insights into how active serpins are stabilized and how conformational changes are induced by protease binding. RESULTS: The 2.1 A structure shows that the RCL of serpin 1K, like that of active alpha1-antitrypsin, is canonical, complimentary and ready to bind to the target protease between P3 and P3 (where P refers to standard protease nomenclature),. In the hinge region (P17-P13), however, the RCL of serpin 1K, like ovalbumin and alpha1-antichymotrypsin, forms tight interactions that stabilize the five-stranded closed form of betasheet A. These interactions are not present in, and are not compatible with, the observed structure of active alpha1-antitrypsin. CONCLUSIONS: Serpin 1K may represent the best resting conformation for serpins - canonical near P1, but stabilized in the closed conformation of betasheet A. By comparison with other active serpins, especially alpha1-antitrypsin, a model is proposed in which interaction with the target protease near P1 leads to conformational changes in betasheet A of the serpin.
Subject(s)
Serpins/chemistry , Amino Acid Sequence , Animals , Computer Simulation , Crystallization , Manduca , Models, Molecular , Molecular Sequence Data , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Mitogen-activated protein (MAP) kinases are activated with great specificity by MAP/ERK kinases (MEKs). The basis for the specific activation is not understood. In this study chimeras composed of two MAP kinases, extracellular signal-regulated protein kinase 2 and p38, were assayed in vitro for phosphorylation and activation by different MEK isoforms to probe the requirements for productive interaction of MAP kinases with MEKs. Experimental results and modeling support the conclusion that the specificity of MEK/MAP kinase phosphorylation results from multiple contacts, including surfaces in both the N- and C-terminal domains.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Enzyme Activation , MAP Kinase Kinase 1 , MAP Kinase Kinase 3 , MAP Kinase Kinase 6 , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase Kinases/metabolism , Models, Molecular , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Recombinant Fusion Proteins/metabolism , p38 Mitogen-Activated Protein KinasesABSTRACT
Fus3p and Kss1p act at the end of a conserved signaling cascade that mediates numerous cellular responses for mating. To determine the role of Fus3p in different outputs, we isolated and characterized a series of partial-function fus3 point mutants for their ability to phosphorylate a substrate (Ste7p), activate Ste12p, undergo G1 arrest, form shmoos, select partners, mate, and recover. All the mutations lie in residues that are conserved among MAP kinases and are predicted to affect either enzyme activity or binding to Ste7p or substrates. The data argue that Fus3p regulates the various outputs assayed through the phosphorylation of multiple substrates. Different levels of Fus3p function are required for individual outputs, with the most function required for shmoo formation, the terminal output. The ability of Fus3p to promote shmoo formation strongly correlates with its ability to promote G1 arrest, suggesting that the two events are coupled. Fus3p promotes recovery through a mechanism that is distinct from its ability to promote G1 arrest and may involve a mechanism that does not require kinase activity. Moreover, catalytically inactive Fus3p inhibits the ability of active Fus3p to activate Ste12p and hastens recovery without blocking G1 arrest or shmoo formation. These results raise the possibility that in the absence of sustained activation of Fus3p, catalytically inactive Fus3p blocks further differentiation by restoring mitotic growth. Finally, suppression analysis argues that Kss1p contributes to the overall pheromone response in a wild-type strain, but that Fus3p is the critical kinase for all of the outputs tested.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Fungal Proteins/metabolism , Mitogen-Activated Protein Kinases , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/physiology , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , G1 Phase , Mating Factor , Mitogen-Activated Protein Kinase Kinases , Models, Molecular , Peptides/pharmacology , Pheromones/pharmacology , Phosphorylation , Point Mutation , Protein Conformation , Protein Kinases/metabolism , Saccharomyces cerevisiae/drug effects , Signal Transduction , Substrate Specificity , Transcriptional ActivationABSTRACT
BACKGROUND: The mitogen-activated protein (MAP) kinases are important signaling molecules that participate in diverse cellular events and are potential targets for intervention in inflammation, cancer, and other diseases. The MAP kinase p38 is responsive to environmental stresses and is involved in the production of cytokines during inflammation. In contrast, the activation of the MAP kinase ERK2 (extracellular-signal-regulated kinase 2) leads to cellular differentiation or proliferation. The anti-inflammatory agent pyridinylimidazole and its analogs (SB [SmithKline Beecham] compounds) are highly potent and selective inhibitors of p38, but not of the closely-related ERK2, or other serine/threonine kinases. Although these compounds are known to bind to the ATP-binding site, the origin of the inhibitory specificity toward p38 is not clear. RESULTS: We report the structural basis for the exceptional selectivity of these SB compounds for p38 over ERK2, as determined by comparative crystallography. In addition, structural data on the origin of olomoucine (a better inhibitor of ERK2) selectivity are presented. The crystal structures of four SB compounds in complex with p38 and of one SB compound and olomoucine in complex with ERK2 are presented here. The SB inhibitors bind in an extended pocket in the active site and are complementary to the open domain structure of the low-activity form of p38. The relatively closed domain structure of ERK2 is able to accommodate the smaller olomoucine. CONCLUSIONS: The unique kinase-inhibitor interactions observed in these complexes originate from amino-acid replacements in the active site and replacements distant from the active site that affect the size of the domain interface. This structural information should facilitate the design of better MAP-kinase inhibitors for the treatment of inflammation and other diseases.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinases , Adenosine Triphosphate/metabolism , Catalytic Domain/drug effects , Cell Differentiation , Cell Division , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Kinetin , Models, Chemical , Models, Molecular , Protein Conformation , Purines/chemistry , Purines/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Pyrimidines/pharmacology , Structure-Activity Relationship , p38 Mitogen-Activated Protein KinasesABSTRACT
Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase compete with ATP for binding. Mutation of 23 residues in the ATP pocket indicated that several residues which affected binding of pyridinyl imidazole photoaffinity cross-linker 125I-SB 206718 did not affect kinase activity, and vice versa, suggesting that pyridinyl imidazoles bind p38 differently than ATP. Two close homologues of p38, SAPK3 and SAPK4, are not inhibited by SB 203580 and differ from p38 by three amino acids near the hinge of the ATP pocket. Substitution of the three amino acids in p38 by those in SAPK3/4 (Thr-106, His-107, and Leu-108 to Met, Pro, and Phe) resulted in decreased 125I-SB 206718 cross-linking and loss of inhibition by SB 203580. Substitution of just Thr-106 by Met resulted in incomplete loss of inhibition. Conversely, substitution of the three amino acids of p38 into SAPK3, SAPK4, or the more distantly related JNK1 resulted in inhibition by SB 203580, whereas mutation of just Met-106 to Thr resulted in weaker inhibition. These results indicate that these three amino acids can confer specificity and sensitivity to SB 203580 for at least two different classes of MAPKs.
Subject(s)
Adenosine Triphosphate/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Mitogen-Activated Protein Kinases , Protein Kinase Inhibitors , Pyridines/pharmacology , Amino Acid Substitution , Binding Sites , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Crystallography, X-Ray , HeLa Cells , Histidine/metabolism , Humans , JNK Mitogen-Activated Protein Kinases , Methionine/metabolism , Mitogen-Activated Protein Kinase 12 , Mitogen-Activated Protein Kinase 13 , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Protein Kinases/genetics , Structure-Activity Relationship , Threonine/metabolism , p38 Mitogen-Activated Protein KinasesABSTRACT
The structure of the active form of the MAP kinase ERK2 has been solved, phosphorylated on a threonine and a tyrosine residue within the phosphorylation lip. The lip is refolded, bringing the phosphothreonine and phosphotyrosine into alignment with surface arginine-rich binding sites. Conformational changes occur in the lip and neighboring structures, including the P+1 site, the MAP kinase insertion, the C-terminal extension, and helix C. Domain rotation and remodeling of the proline-directed P+1 specificity pocket account for the activation. The conformation of the P+1 pocket is similar to a second proline-directed kinase, CDK2-CyclinA, thus permitting the origin of this specificity to be defined. Conformational changes outside the lip provide loci at which the state of phosphorylation can be felt by other cellular components.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Binding Sites , Crystallography , Dimerization , Enzyme Activation , Mitogen-Activated Protein Kinase 1 , Molecular Sequence Data , Phosphorylation , Proline/chemistry , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Substrate Specificity , Threonine/metabolism , Tyrosine/metabolismABSTRACT
The structure of mitogen-activated protein (MAP) kinase p38 has been solved at 2.1-A to an R factor of 21.0%, making p38 the second low activity MAP kinase solved to date. Although p38 is topologically similar to the MAP kinase ERK2, the phosphorylation Lip (a regulatory loop near the active site) adopts a different fold in p38. The peptide substrate binding site and the ATP binding site are also different from those of ERK2. The results explain why MAP kinases are specific for different activating enzymes, substrates, and inhibitors. A model presented for substrate and activator interactions has implications for the evolution of protein kinase cascades.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Mitogen-Activated Protein Kinases , Protein Conformation , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinases/biosynthesis , Calcium-Calmodulin-Dependent Protein Kinases/isolation & purification , Computer Simulation , Crystallography, X-Ray , Humans , Mice , Mitogen-Activated Protein Kinase 1 , Models, Molecular , Models, Structural , Molecular Sequence Data , Protein Structure, Secondary , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid , Software , p38 Mitogen-Activated Protein KinasesABSTRACT
The serpin plasminogen activator inhibitor 1 (PAI-1) folds into an active structure and then converts slowly to a more stable, but low-activity, "latent" conformation [Hekman, C. M., & Loskutoff, D. J. (1985) J. Biol. Chem. 260, 11581-11587]. Thus, the folding of PAI-1 is apparently under kinetic control. We have determined the urea denaturation and refolding transitions of both latent and active PAI-1 proteins by using intrinsic tryptophan fluorescence. While folding of active PAI-1 is reversible, the denaturation and refolding of latent PAI-1 are not. Instead, denatured latent PAI-1 refolds in lower concentrations of urea to give the active protein. Thus, the high-stability latent conformation is kinetically inaccessible over a range of urea concentrations. Complete denaturation of latent PAI-1 occurs at 5.5 M urea [delta G(H2O) approximately 21 kcal] whereas active PAI-1 denatures in only 3.8 M urea [delta G(H2O) approximately 12 kcal]. The fluorescence emission profile, as a function of urea of both the active and latent forms of the protein, reveals intermediates with partial structure. Circular dichroism measurements and limited protease digestion with Lys-C suggest that the intermediate in the denaturation of latent PAI-1 retains most of the secondary structure of the fully folded protein, whereas the intermediate in the denaturation of active PAI-1 exhibits significant loss of secondary structure. The Lys-C digestion patterns show that the active protein is more susceptible to proteolysis near sheet A than is the latent form. The studies suggest a model for the kinetically controlled folding pathway of PAI-1.
Subject(s)
Plasminogen Activator Inhibitor 1/chemistry , Binding Sites , Circular Dichroism , Humans , In Vitro Techniques , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activator Inhibitor 1/metabolism , Protein Conformation , Protein Denaturation , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , ThermodynamicsABSTRACT
In chemomechanical energy transducing assemblies such as muscle and ATP synthase, substrates and macromolecules are locked together as partners where energy available from (or required for) a chemical transformation is exchanged with protein conformational changes. Allosteric binding proteins and enzymes are also chemomechanical energy transducers, using binding energy to generate protein conformational changes, and transduce energy in amounts almost as large as those used to drive muscle contraction and the synthesis of ATP. The recently determined structure of the F1-ATPase reveals a direct correspondence between the types of conformational changes in this transducer and simpler allosteric binding proteins and enzymes. Therefore, we can examine the structural and energetic data available on allosteric proteins to understand the linkage between ligand binding and global conformational changes in more complex energy transducing assemblies.
Subject(s)
Energy Metabolism , Proton-Translocating ATPases/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation , Animals , Catalysis , Protein Conformation , Proton-Translocating ATPases/chemistryABSTRACT
Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood. We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2. The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates. The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat. Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants. The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2. ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme. The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis.
Subject(s)
Adenosine Triphosphate/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Point Mutation , Amino Acid Sequence , Binding Sites/genetics , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Conserved Sequence , DNA-Binding Proteins/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Kinetics , Lysine/chemistry , Lysine/genetics , Mitogen-Activated Protein Kinase 1 , Models, Molecular , Molecular Sequence Data , Molecular Structure , Mutagenesis, Site-Directed , Myelin Basic Protein/metabolism , Neoplasm Proteins/metabolism , Peptides/genetics , Peptides/metabolism , Protein Conformation , Substrate SpecificityABSTRACT
Trypanosoma brucei ornithine decarboxylase, expressed and purified from E. coli, has been crystallized by the vapor diffusion method using PEG 3350 as a precipitant. The crystals belong to the monoclinic space group P2(1) and have cell constants of a = 66.3 angstroms, b = 151.8 angstroms, c = 83.7 angstroms, and beta = 101.2 degrees. While larger crystals are twinned, smaller crystals (0.4 x 0.3 x 0.05 mm3) are single.
Subject(s)
Ornithine Decarboxylase/chemistry , Trypanosoma brucei brucei/enzymology , Animals , Crystallography, X-Ray , Escherichia coli/genetics , Ornithine Decarboxylase/genetics , Recombinant Proteins/chemistry , Trypanosoma brucei brucei/geneticsABSTRACT
We used sequence and structural comparisons to determine the fold for eukaryotic ornithine decarboxylase, which we found is related to alanine racemase. These enzymes have no detectable sequence identity with any protein of known structure, including three pyridoxal phosphate-utilizing enzymes. Our studies suggest that the N-terminal domain of ornithine decarboxylase folds into a beta/alpha-barrel. Through the analysis of known barrel structures we developed a topographic model of the pyridoxal phosphate-binding domain of ornithine decarboxylase, which predicts that the Schiff base lysine and a conserved glycine-rich sequence both map to the C-termini of the beta-strands. Other residues in this domain that are likely to have essential roles in catalysis, substrate, and cofactor binding were also identified, suggesting that this model will be a suitable guide to mutagenic analysis of the enzyme mechanism.