Abstract: A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKβ. In this Letter we document our efforts at further optimization of this series, culminating in 2 with submicromolar potency in a HWB assay and efficacy in a CIA mouse model.
Abstract: A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKβ. In this Letter we document our early efforts at optimization of the quinoline core, the imidazole and the semithiocarbazone moiety. Most potency gains came from substitution around the 6- and 7-positions of the quinoline ring. Replacement of the semithiocarbazone with a semicarbazone decreased potency but led to some measurable exposure.
Abstract: Bruton's tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X-ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly-rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2-kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 3(10)- and α-helices, one of which collapses into the adenosine-5'-triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG-out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure-based design of specific inhibitors.
Abstract: JNK2 and p38alpha are closely related mitogen-activated protein kinases that regulate various cellular activities and are considered drug targets for inflammatory diseases. We have determined the X-ray crystal structure of the clinical phase II p38alpha inhibitor BIRB796 bound to its off-target JNK2. This shows for the first time a JNK subfamily member in the DFG-out conformation. The fully resolved activation loop reveals that BIRB796 inhibits JNK2 activation by stabilizing the loop in a position that does not allow its phosphorylation by upstream kinases. The structure suggests that substituents at the BIRB796 morpholino group and modifications of the t-butyl moiety should further increase the p38alpha to JNK2 potency ratio. For the design of selective DFG-out binding JNK2 inhibitors, the binding pocket of the BIRB796 tolyl group may have the best potential.
Abstract: Spleen tyrosine kinase is considered an attractive drug target for the treatment of allergic and antibody mediated autoimmune diseases. We have determined the co-crystal structures of spleen tyrosine kinase complexed with three known inhibitors: YM193306, a 7-azaindole derivative and R406. The cis-cyclohexyldiamino moiety of YM193306 is forming four hydrophobically shielded polar interactions with the spleen tyrosine kinase protein and is therefore crucial for the high potency of this inhibitor. Its primary amino group is inducing a conformational change of the spleen tyrosine kinase DFG Asp side chain. The crystal structure of the 7-azaindole derivative bound to spleen tyrosine kinase is the first demonstration of a 2-substituted 7-azaindole bound to a protein kinase. Its indole-amide substituent is tightly packed between the N- and C-terminal kinase lobes. The co-crystal structure of the spleen tyrosine kinase-R406 complex shows two main differences to the previously reported structure of spleen tyrosine kinase soaked with R406: (i) the side chain of the highly conserved Lys is disordered and not forming a hydrogen bond to R406 and (ii) the DFG Asp side chain is pointing away from and not towards R406. The novel protein-ligand interactions and protein conformational changes revealed in these structures guide the rational design and structure-based optimization of second-generation spleen tyrosine kinase inhibitors.
Abstract: Promiscuous binders achieve enzyme inhibition using a nonspecific aggregation-type binding mechanism to proteins. These compounds are a source of false-positive hits in biochemical inhibition assays and should be removed from screening hit lists because they are not good candidates to initiate medicinal chemistry programs. We introduce a robust approach to identify these molecules early in the lead generation process using real time surface plasmon resonance based biosensors to observe the behavior of the binding interactions between promiscuous compounds and proteins. Furthermore, the time resolution of the assay reveals a number of distinct mechanisms that promiscuous compounds employ to inhibit enzyme function and indicate that the type of mechanism can vary depending on the protein target. A classification scheme for these compounds is presented that can be used to rapidly characterize the hits from high-throughput screens and eliminate compounds with a nonspecific mechanism of inhibition.
Abstract: c-Jun N-terminal kinase (JNK) 2 is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. MAP kinases share a common sequence insertion called "MAP kinase insert", which, for ERK2, has been shown to interact with regulatory proteins and, for p38alpha, has been proposed to be involved in the regulation of catalytic activity. We have determined the crystal structure of human JNK2 complexed with an indazole inhibitor by applying a high-throughput protein engineering and surface-site mutagenesis approach. A novel conformation of the activation loop is observed, which is not compatible with its phosphorylation by upstream kinases. This activation inhibitory conformation of JNK2 is stabilized by the MAP kinase insert that interacts with the activation loop in an induced-fit manner. We therefore suggest that the MAP kinase insert of JNK2 plays a role in the regulation of JNK2 activation, possibly by interacting with intracellular binding partners.
Abstract: Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with approximately 1-10mM binding affinity (K(D)) were iteratively optimized to give leads with approximately 200nM biochemical activity and low microM cellular activity in a Replicon assay.
Abstract: IL-1R-associated kinase (IRAK)4 plays a central role in innate and adaptive immunity, and is a crucial component in IL-1/TLR signaling. We have determined the crystal structures of the apo and ligand-bound forms of human IRAK4 kinase domain. These structures reveal several features that provide opportunities for the design of selective IRAK4 inhibitors. The N-terminal lobe of the IRAK4 kinase domain is structurally distinctive due to a loop insertion after an extended N-terminal helix. The gatekeeper residue is a tyrosine, a unique feature of the IRAK family. The IRAK4 structures also provide insights into the regulation of its activity. In the apo structure, two conformations coexist, differing in the relative orientation of the two kinase lobes and the position of helix C. In the presence of an ATP analog only one conformation is observed, indicating that this is the active conformation.
Abstract: A novel class of highly selective inhibitors of p38 MAP kinase was discovered from high throughput screening. The synthesis and optimization of a series of 5-amino-N-phenyl-1H-pyrazol-4-yl-3-phenylmethanones is described. An X-ray crystal structure of this series bound in the ATP binding pocket of unphosphorylated p38alpha established the presence of a unique hydrogen bond between the exocyclic amine of the inhibitor and threonine 106 which likely contributes to the selectivity for p38. The crystallographic information was used to optimize the potency and physicochemical properties of the series. The incorporation of the 2,3-dihydroxypropoxy moiety on the pyrazole scaffold resulted in a compound with excellent drug-like properties including high oral bioavailability. These efforts identified 63 (RO3201195) as an orally bioavailable and highly selective inhibitor of p38 which was selected for advancement into Phase I clinical trials.
Abstract: Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV) polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb- and a palm-binding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.
Abstract: The introduction of 3-arylmethyl, 3-aryloxy and 3-arylthio moieties into a 6-methylsulfonylindole framework using rational drug design led to potent, selective COX-2 inhibitors having efficacy in a rat carrageenan air pouch model. Incorporation of a conformationally more rigid 3-aroyloxy substituent onto the 6-methylsulfonylindole scaffold led to selective, but considerably less potent COX-2 inhibitors. Variation of the hydrophilicity and size of the indole 2-substituent of 3-arylthio-6-methylsulfonylindole inhibitors led to modulation of the COX-2 human whole blood (HWB) potency and selectivity.
Abstract: Inhibition of the biosynthesis of proinflammatory cytokines such as tumor necrosis factor and interleukin-1 via p38 has been an approach toward the development of a disease modifying agent for the treatment of chronic inflammation and autoimmune diseases. The development of a new core structure of p38 inhibitors, 3-(4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo[3,2-b] pyridine, is described. X-ray crystallographic data of the lead bound to the active site of p38 was used to guide the optimization of the series. Specific focus was placed on modulating the physical properties of the core while maintaining potent inhibition of p38. These efforts identified 42c as a potent inhibitor of p38, which also possessed the required physical properties worthy of advanced studies.
Abstract: There is significant demand to rapidly obtain protein structure information for both structural genomics and drug discovery applications. To meet this demand, all steps in the process of determining protein structure by X-ray crystallography need to be optimized and streamlined with high-throughput methodologies. This communication describes a method that brings high-throughput technology to protein crystallization in both manual and automated modes, suitable for virtually every crystallography laboratory.
Abstract: Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15-lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining the oxygenation specificity of 5-, 12-, and 15-lipoxygenases. Based on the x-ray crystal structure of rabbit 15-lipoxygenase, Ile(593) appeared to be important in defining size and shape of the substrate-binding site in 15-lipoxygenases. We found that substitution of Ile(593) with alanine shifted the positional specificity of this enzyme toward 12-lipoxygenation. To compare the importance of position 593 with previously defined determinants for the oxygenation specificity, we introduced small (alanine-scan) or large amino acids (phenylalanine-scan) at critical positions surrounding the putative fatty acid-binding site, so that the volume of the pocket was either increased or decreased. Enlargement or alteration in packing density within the substrate binding pocket in the rabbit 15-lipoxygenase increased the share of 12-lipoxygenase products, whereas a smaller active site favored 15-lipoxygenation. Simultaneous substitution of both large and small residues in the context of either a 15- or 12-lipoxygenase indicated that there is a functional interplay of the sequence determinants for lipoxygenation specificity. If the 15-lipoxygenase active site is enlarged excessively, however, no lipoxygenation was observed anymore. Together these results indicate the importance of the overall size and shape of the arachidonic acid binding pocket in defining the specificity of lipoxygenase reaction.
Abstract: The X-ray crystal structures of the catalytic domain of human collagenase-3 (MMP-13) and collagenase-1 (MMP-1) with bound inhibitors provides a basis for understanding the selectivity profile of a novel series of matrix metalloprotease (MMP) inhibitors. Differences in the relative size and shape of the MMP S1' pockets suggest that this pocket is a critical determinant of MMP inhibitor selectivity. The collagenase-3 S1' pocket is long and open, easily accommodating large P1' groups, such as diphenylether. In contrast, the collagenase-1 S1' pocket must undergo a conformational change to accommodate comparable P1' groups. The selectivity of the diphenylether series of inhibitors for collagenase-3 is largely determined by their affinity for the preformed S1' pocket of collagenase-3, as compared to the induced fit in collagenase-1.
Abstract: Here we report the first structure of a mammalian 15-lipoxygenase. The protein is composed of two domains; a catalytic domain and a previously unrecognized beta-barrel domain. The N-terminal beta-barrel domain has topological and sequence identify to a domain in the mammalian lipases, suggesting that these domains may have similar functions in vivo. Within the C-terminal domain, the lipoxygenase substrate binding site is a hydrophobic pocket defined by a bound inhibitor. Arachidonic acid can be docked into this deep hydrophobic pocket with the methyl end extending down into the bottom of the pocket and the acid end tethered by a conserved basic residue on the surface of the enzyme. This structure provides a unifying hypothesis for the positional specificity of mammalian lipoxygenases.
Abstract: Phosphorylation of glycogen phosphorylase at residue Ser14 triggers a conformational transition that activates the enzyme. The N-terminus of the protein, in response to phosphorylation, folds into a 310 helix and moves from its location near a cluster of acidic residues on the protein surface to a site at the dimer interface where a pair of arginine residues form charged hydrogen bonds with the phosphoserine. Site-directed mutagenesis was used to replace Ser14 with Asp and Glu residues, analogs of the phosphoserine, that might be expected to participate in ionic interactions with the arginine side chains at the dimer interface. Kinetic analysis of the mutants indicates that substitution of an acidic residue in place of Ser14 at the site of regulatory phosphorylation partially activates the enzyme. The S14D mutant shows a 1.6-fold increase in Vmax, a 10-fold decrease in the apparent dissociation constant for AMP, and a 3-fold decrease in the S0.5 for glucose 1-phosphate. The S14E mutant behaves similarly, showing a 2.2-fold increase in Vmax, a 6-fold decrease in the apparent dissociation constant for AMP, and a 2-fold decrease in the S0.5 for glucose 1-phosphate. The ability of the mutations to enhance binding of AMP and glucose 1-phosphate and to raise catalytic activity suggests that the introduction of a carboxylate side chain at position 14 promotes docking of the N-terminus at the subunit interface and concomitant stabilization of the activated conformation of the enzyme. Like the native enzyme, both mutants show significant activity only in the presence of the activator, AMP. Full activation, analogous to that provided by covalent phosphorylation of the enzyme, likely is not achieved because of differences in the charge and the geometry of ionic interactions at the phosphorylation site.
Abstract: The first crystal structure of human cyclooxygenase-2, in the presence of a selective inhibitor, is similar to that of cyclooxygenase-1. The structure of the NSAID binding site is also well conserved, although there are differences in its overall size and shape which may be exploited for the further development of selective COX-2 inhibitors. A second COX-2 structure with a different bound inhibitor displays a new, open conformation at the bottom of the NSAID binding site, without significant changes in other regions of the COX-2 structure. These two COX-2 structures provide evidence for the flexible nature of cyclooxygenase, revealing details about how substrate and inhibitor may gain access to the cyclooxygenase active site from within the membrane.
Abstract: Matrilysin (MAT) prefers leucine over residues that have aromatic side chains at the P1' position of peptide and protein substrates, while stromelysin (HFS) has a broader specificity. The X-ray structures of these enzymes show that their respective S1' subsites differ primarily due to the amino acids present at positions 214 and 215. To examine the role that these residues play in determining P1' specificity, the amino acids at these positions in matrilysin have been replaced by those found in stromelysin (MAT: Y214L, MAT:A215V, and MAT:Y214L/A215V). The specificity and activity of MAT:A215V are similar to those of wild type matrilysin. Both MAT:Y214L and MAT:Y214L/A215V, however, have P1' specificities that are more similar to stromelysin than matrilysin. Specifically, these enzymes exhibit an 8- to 9-fold reduction in kcat/KM toward a peptide substrate with Leu in subsite P1' relative to wild type matrilysin. This is predominantly the result of an approximate 5-fold decrease in kcat. The KM values only partially increase toward the value observed for stromelysin. Studies of the pre-steady-state reaction of wild type and mutant matrilysin with substrates with Leu and Tyr residues in the P1' position confirm that the KM values for these reactions reflect KD values for substrate binding. Thus, replacement of a single tyrosine residue in the S1' pocket of matrilysin by leucine alters its P1' specificity to resemble that of stromelysin. In contrast, alteration of the S1' subsite of stromelysin (HFS:L214Y/V215A) to resemble matrilysin increases activity (i.e., higher kcat/KM) toward peptide substrates with both leucine and residues with aromatic side chains in the P1' position with only a partial increase in specificity for Leu. These increases in activity are the result of decreases in the KM values for these reactions.
Abstract: Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory disorders and are, therefore, important targets for drug discovery. Both plant and mammalian lipoxygenases catalyze the dioxygenation of polyunsaturated fatty acids, which contain one or more 1,4-cis,cis-pentadiene units to yield hydroperoxide products. At the time this study was initiated, soybean lipoxygenase-1 was the only lipoxygenase for which an atomic resolution structure had been determined. No structure of lipoxygenase with substrate or inhibitor bound is currently available. A model of arachidonic acid docked into the proposed substrate binding site in the soybean structure is presented here. Analysis of this model suggested two residues, an aromatic residue and a positively charged residue, could be critical for substrate binding. Validation of this model is provided by site-directed mutagenesis of human 15-lipoxygenase, despite the low amino acid sequence identity between the soybean and mammalian enzymes. Both a positively charged amino acid residue (Arg402) and an aromatic amino acid residue (Phe414) are identified as critical for the binding of fatty acid substrates in human 15-lipoxygenase. Thus, binding determinants shown to be characteristic of non-enzymatic fatty acid-binding proteins are now implicated in the substrate binding pocket of lipoxygenases.
Abstract: Human promatrilysin (matrix metalloproteinase-7) has been produced in Escherichia coli as an N-terminal fusion protein with ubiquitin. The insoluble product was solubilized, refolded, and activated with amino-phenylmercuric acetate. Activation of the fusion protein demonstrated kinetics and intermediates that were very similar to those observed during activation of promatrilysin produced in Chinese Hamster Ovary (CHO) cells. Following activation, matrilysin was purified to > 95% homogeneity using a Sepharose-Pro-Leu-Gly-NHOH affinity column. The matrilysin purified by this procedure is indistinguishable from the enzyme purified from CHO cells with respect to the kinetic parameters for hydrolysis of a peptide substrate and the ability to obtain diffraction quality crystals in the presence of an inhibitor of the enzyme. Additionally, to facilitate detailed kinetic analyses of matrilysin, a new fluorogenic peptide substrate with the optimized sequence Dnp-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (Dnp, dinitrophenyl) has been synthesized. This peptide is the best substrate developed for matrilysin thus far with Km and kcat values of 26 microM and 5.0 s-1, respectively.
Abstract: Matrix metalloproteases are a family of enzymes that play critical roles in the physiological and pathological degradation of the extracellular matrix. These enzymes may be important therapeutic targets for the treatment of various diseases where tissue degradation is part of the pathology, such as cancer and arthritis. Matrilysin is the smallest member of this family of enzymes, all of which require zinc for catalytic activity. The first X-ray crystal structures of human matrilysin are presented. Inhibitors of metalloproteases are often characterized by the chemical group that interacts with the active site zinc of the protein. The structures of matrilysin complexed with hydroxamate (maximum resolution 1.9 A), carboxylate (maximum resolution 2.4 A), and sulfodiimine (maximum resolution 2.3 A) inhibitors are presented here and provide detailed information about how each functional group interacts with the catalytic zinc. Only the zinc-coordination group is variable in this series of inhibitors. Examination of these inhibitor-matrilysin complexes emphasizes the dominant role the zinc-coordinating group plays in determining the relative potencies of the inhibitors. The structures of these matrilysin-inhibitor complexes also provide a basis for comparing the catalytic mechanism of MMPs and other metalloproteins.
Abstract: Activation of protein function through phosphorylation can be mimicked by the engineering of specific metal binding sites. The addition of two histidine residues to glycogen phosphorylase allows enzymatic activation by transition metals in a cooperative and allosteric manner. Crystal structures of the metallo-enzyme have been determined and show that the structural transition induced upon metal binding (Ni2+) is, in part, analogous to the mode of activation of the native enzyme. The designed metal activation site allows assignment of the structural changes which trigger activation in this allosteric enzyme and, further, provide insight into the evolutionary development of multiple activation sites.
Abstract: Mammalian phosphorylase isozymes from muscle, brain and liver were expressed in Escherichia coli and purified from the crude bacterial cell extracts in one step using a copper-loaded, metal-affinity matrix. Good chromatographic behavior, enzyme activity and protein stability were maintained by judicious choice of pH and buffer which contained 250 mM sodium chloride and 25 mM beta-glycerophosphate at pH 7.0. Small amounts of beta-mercaptoethanol and EDTA in the buffers further stabilized the enzymes, but stripped some of the metal from the column which, nonetheless, retained good chromatographic characteristics. Owing to the presence of multiple surface histidine residues in the phosphorylase dimers, good enzyme purities (90-98%) and recoveries (>90%) were routinely obtained from crude bacterial lysates after two passes through the copper column. Of the various metal ions which were investigated, Cu2+ gave the best chromatographic results. Imidazole gradients at constant pH were used to selectively desorb the phosphorylase from the metal column whose capacity for phosphorylase binding in the presence of bacterial proteins exceeded 30 mg enzyme per milliliter of matrix.
Abstract: The catalytic subunit of rabbit skeletal muscle protein phosphatase-1 was expressed in Escherichia coli. Expression of phosphatase-1 in the pET3a vector, which is based on the use of the T7 promoter, resulted in the expression of the enzyme as an insoluble aggregate. The insoluble enzyme could be renatured by high dilutions of the urea-solubilized protein in buffers containing dithiothreitol, Mn2+, and high NaCl concentrations. However, under all conditions tested, only partial (less than 5%) renaturation was achieved. A second attempt was made using a vector with the trp-lac hybrid promoter. In this case it was possible to express the enzyme as a soluble protein at levels of 3-4% of the soluble E. coli protein. The recombinant enzyme was purified by DEAE-Sepharose and heparin-Sepharose chromatography. Approximately 20 mg of purified enzyme was reproducibly obtained from the cells derived from 2 liters of culture. The purified enzyme had a specific activity toward phosphorylase alpha comparable to that reported for the authentic protein and had an Mr of 37,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The recombinant enzyme displayed similar sensitivities to inhibition by inhibitor-2, okadaic acid, and microcystin-LR as for the protein isolated from rabbit muscle. At all stages of purification the recombinant phosphatase behaved as an essentially inactive enzyme that required the presence of microM Mn2+ for full expression of its activity.
Abstract: A transducer is a device that receives energy from one system and transmits it, often in a different form, to another. Glycogen phosphorylase receives information from the cell or organism in the form of metabolic signals. The energy associated with the binding of these ligand signals is integrated and transmitted at an atomic level, allowing precise adjustment of the enzymatic activity. Understanding this elegant allosteric control has required several different approaches, but the structural requirements of allostery are being defined.
Abstract: Muscle and liver glycogen phosphorylase isozymes differ in their responsiveness to the activating ligand AMP. The muscle enzyme, which supplies glucose in response to strenuous activity, binds AMP cooperatively, and its enzymatic activity becomes greatly enhanced. The liver isozyme regulates the level of blood glucose, and AMP is not the primary activator. In muscle glycogen phosphorylase, the residue proline 48 links two secondary structural elements that bind AMP. This amino acid residue is replaced with a threonine in the liver isozyme; unlike the muscle enzyme, liver binds AMP noncooperatively, and the enzymatic activity is not greatly increased. We have substituted proline 48 in the muscle enzyme with threonine, alanine, and glycine and characterized the recombinant enzymes kinetically and structurally to determine if proline at this position is critical for cooperative AMP binding and activation. Importantly, all of the engineered enzymes were fully activated by phosphorylation, indicating that enzymatic activity was not compromised. Only the mutant enzyme with alanine at position 48 responds like the wild-type enzyme to the presence of AMP, indicating that proline is not absolutely required for full cooperative activation. The substitution of either threonine or glycine at this position, however, creates enzymes that no longer bind AMP cooperatively. The enzyme with threonine at position 48 further mimics the liver enzyme, in that the maximal enzymatic activity is also reduced. Significantly, the glycine substitution caused the enzyme to be fully activated by AMP, although binding was not cooperative. The hyperactivation of the glycine mutant by AMP suggests that the total free energy of activation has decreased.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: An intrinsic molecular property of a protein domain can be determined by calculating its principal axes from the inertia tensor matrix. The mass-weighted principal axes can be used to calculate an ellipsoid representing the shape of the protein domain, providing an easy means of visualizing domain movements. Most importantly, the mass-weighted principal axes provide an intuitive means of characterizing domain relationships within a protein, as well as the disposition of domains in different protein conformers. Thus, this method provides a simple, quantitative description of differences of domain positions within various protein structures. We show the utility of this method by characterizing the quaternary and tertiary differences as observed in eight structures of phosphorylated or dephosphorylated glycogen phosphorylase with different effectors bound. This analysis revealed domain movements which were characteristic of the activated phosphorylase structures. The monomers of the phosphorylase dimer were found to move apart by a 2.5-A translation and to rotate apart, in three orthogonal directions, by a minimum of 3.2 degrees. Analysis of the three domains within the phosphorylase monomer showed that both simple and complex domain movements occur and that multiple domain configurations are energetically stable. We suggest that the C-terminal domain of phosphorylase moves along a simple path in the transition from an inactive to active conformation. The direction of translation and rotation is consistent, but the magnitude is variable. In contrast, this analysis showed that the activation domain did not behave as a rigid body, and therefore, the motion of this domain is not as easily characterized.
Abstract: Liver and muscle glycogen phosphorylases, which are products of distinct genes, are both activated by covalent phosphorylation, but in the unphosphorylated (b) state, only the muscle isozyme is efficiently activated by the allosteric activator AMP. The different responsiveness of the phosphorylase isozymes to allosteric ligands is important for the maintenance of tissue and whole body glucose homeostasis. In an attempt to understand the structural determinants of differential sensitivity of the muscle and liver isozymes to AMP, we have developed a bacterial expression system for the liver enzyme, allowing native and engineered proteins to be expressed and characterized. Engineering of the single amino acid substitutions Thr48Pro, Met197Thr and the double mutant Thr48Pro, Met197Thr in liver phosphorylase, and Pro48Thr in muscle phosphorylase, did not qualitatively change the response of the two isozymes to AMP. These sites had previously been implicated in the configuration of the AMP binding site. However, when nine amino acids among the first 48 in liver phosphorylase were replaced with the corresponding muscle phosphorylase residues (L1M2-48L49-846), the engineered liver enzyme was activated by AMP to a higher maximal activity than native liver phosphorylase. Interestingly, the homotropic cooperativity of AMP binding was unchanged in the engineered phosphorylase b protein, and heterotropic cooperativity between the glucose-1-phosphate and AMP sites was only slightly enhanced. The native liver, native muscle and L1M2-48L49-846 phosphorylases were converted to the a form by treatment with purified phosphorylase kinase; the maximal activity of the chimeric a enzyme was greater than the native liver a enzyme and approached that of muscle phosphorylase a. From these results we suggest that tissue-specific phosphorylase isozymes have evolved a complex mechanism in which the N-terminal 48 amino acids modulate intrinsic activity (Vmax), probably by affecting subunit interactions, and other, as yet undefined regions specify the allosteric interactions with ligands and substrates.
Abstract: We have expressed the serine protease inhibitor ecotin to high levels (greater than 400 mg/l of cell culture) in its natural mileau, the Escherichia coli periplasm, using the endogenous signal peptide and the heterologous tac promoter. After induction, functional, soluble ecotin comprises 15% of total cellular protein. This expression system has facilitated initiation of a crystallographic study to determine the structural basis for inhibition of the pancreatic serine proteases by ecotin. Ecotin was co-crystallized with rat trypsin mutant D102N. Preliminary crystallographic analysis of co-crystals showed that they diffract to at least 2.7 A, and indicate that they belong to the monoclinic space group, P21. The cell constants are a = 52.0 A, b = 93.3 A, c = 160.7 A, and beta = 96 degrees. Four molecules each of trypsin and ecotin are found in the asymmetric unit.
Abstract: In order to understand how allosteric switches regulate both the catalytic activity and molecular interactions of glycogen phosphorylase, it is necessary to design and analyze variant proteins that test hypotheses about the structural details of the allosteric mechanism. Essential to such an investigation is the ability to obtain large amounts of variant proteins. We developed a system for obtaining milligram amounts (greater than 20 mg/l) of rabbit muscle phosphorylase from bacteria. Phosphorylase aggregates as inactive protein when a strong bacterial promoter is used under full inducing conditions and normal growth conditions. However, when the growth temperature of bacteria expressing phosphorylase is reduced to 22 degrees C we obtain active muscle phosphorylase. The degree to which the induced expression of phosphorylase protein is temperature sensitive depends on the strain of bacteria used. New assay and purification methods were developed to allow rapid purification of engineered phosphorylase proteins from bacterial cultures. The rabbit muscle phosphorylase obtained from the bacterial expression system is enzymatically identical to the enzyme purified from rabbit muscle. The expressed protein crystallizes in the same conditions used for growing crystals of protein from rabbit muscle and the crystal form is isomorphous. Rabbit muscle phosphorylase is one of the largest oligomeric mammalian enzymes successfully expressed in Escherichia coli. Our results indicate that optimization of a combination of growth and induction conditions will be important in the expression of other heterologous proteins in bacteria.
Abstract: We report a new purification of rabbit reticulocyte 15-lipoxygenase that has resulted in the first crystallization of a mammalian lipoxygenase. The enzyme was purified to homogeneity (greater than 98% pure by SDS-PAGE) using high pressure liquid chromatography on hydrophobic-interaction, hydroxyapatite and cation-exchange columns. Crystals were grown by the vapor diffusion method from concentrated solutions of the protein in sodium phosphate buffer, pH 7.0. The hexagonal, rod-shaped crystals were on average 0.09 mm x 0.09 mm x 0.4 mm, with approximate unit cell dimensions of a = b = 260 A, c = 145 A. The crystals diffract to 5 A resolution.
Abstract: The complete amino acid sequence of rabbit skeletal muscle glycogen synthase was deduced from cDNA clones with a composite length of 3317 bp. An mRNA of 3.6 kb was identified by Northern blot analysis of rabbit skeletal muscle RNA. The mRNA coded for a protein of 734 residues with a molecular weight of 83,480. The deduced NH2-terminal and COOH-terminal sequences corresponded to those reported for the purified protein, indicating the absence of any proteolytic processing. At the nucleotide level, the 5' untranslated and coding regions were 79 and 90% identical for rabbit and human muscle glycogen synthases, whereas the 3' untranslated regions were significantly less similar. The enzymes had 97% amino acid sequence identity. Interestingly, the NH2 and COOH termini of rabbit and human muscle glycogen synthase, the regions of phosphorylation, showed the greatest sequence variation (15 of 19 mismatches and two insertion/deletion events), which may indicate different evolutionary constraints in the regulatory and catalytic regions of the molecule.
Abstract: The cDNA for human muscle glycogen synthase encodes a protein of 737 amino acids. The primary structure of glycogen synthase is not related either to bacterial glycogen synthase or to any glycogen phosphorylase. All nine of the serines that are phosphorylated in the rabbit muscle enzyme in vivo are conserved in the human muscle sequence. The amino- and carboxyl-terminal fragments, which contain all the phosphorylation sites, are very negatively charged. Overall the unphosphorylated protein has a charge of -13, while the fully phosphorylated inactive protein has a net charge of -31. The importance of the asymmetrical charge distribution is discussed.
Abstract: We have cloned and sequenced cDNAs encoding the alpha-subunit of rat liver propionyl-CoA carboxylase (PCC), a biotin-dependent, mitochondrial matrix protein. The full-length cDNA spans 3327 base pairs, has a long (895 base pairs) 5'-untranslated region, and encodes a protein of 721 amino acids. In vitro transcription and translation of the full-length rat alpha-PCC cDNA produces a product which is immunoprecipitable with antibodies specific to PCC and has the same apparent molecular weight as in vivo synthesized alpha-PCC. Rat liver alpha-PCC precursor is not quantitatively biotinylated when synthesized in a cell-free rabbit reticulocyte system. Nevertheless, when incubated with isolated rat liver mitochondria in vitro, the precursor is imported and proteolytically cleaved to its mature form. A sequence comparison of rat liver alpha-PCC and other biotinylated polypeptides reveals the absolute conservation of three glycines and one valine in the region surrounding the biotinylated lysine residue. This pattern of conserved residues is also present in lipoylated proteins, where lipoic acid is similarly, covalently attached to a lysine residue. A possible functional role for the conserved glycine residues and further sequence similarity surrounding the site of biotinylation and lipoylation is discussed.
Abstract: Chemical modification of unpaired bases is demonstrated in this study to be a reliable method for determining the conformation of nucleotides in mRNA. The modified nucleotides are identified by primer extension using reverse transcriptase. We have used this procedure to compare the structure of limited regions of SV40 T-antigen mRNA in solution, in nonpolysome-bound cytoplasmic messenger ribonucleoprotein particles, and in nuclear ribonucleoprotein complexes. The results indicate that SV40 T-antigen mRNA adopts a specific structure both in solution and when complexed with cellular proteins. The structures adopted by the mRNA in solution and in native cellular protein particles are very similar.
Abstract: One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.
Abstract: A model for the secondary structure of mouse beta Maj globin messenger RNA is presented based on enzymatic digestion data, comparative sequence and computer analysis. Using 5'-32P-end-labeled beta globin mRNA as a substrate, single-stranded regions were determined with S1 and T1 nucleases and double-stranded regions with V1 ribonuclease from cobra venom. The structure data obtained for ca. 75% of the molecule was introduced into a computer algorithm which predicts secondary structures of minimum free energy consistent with the enzymatic data. Two prominent base paired regions independently derived by phylogenetic analysis were also present in the computer generated structure lending support for the model. An interesting feature of the model is the presence of long-range base pairing interactions which permit the beta globin mRNA to fold back on itself, thereby bringing the 5'- and 3'-noncoding regions within close proximity. This feature is consistent with data from other laboratories suggesting an interaction of the 5'- and 3'-domains in the mammalian globin mRNAs.
Abstract: Analysis of phylogenetically conserved secondary structure has been important in the development of models for the secondary structure of structural RNAs. In this paper, we apply this type of analysis to several families of informational RNAs to evaluate its usefulness in developing secondary structure models for mRNAs and mRNA precursors. We observed many conserved helices in all mRNA groups analyzed. Three criteria were used to identify potential helices which were not conserved solely because of coding sequence constraints, and may therefore be important for the structure and function of the RNA. These results suggest that this approach will be useful in deriving secondary structure models for informational RNAs when used in conjunction with other complementary techniques, and in designing experiments to determine the functional significance of conserved base pairing interactions.
Abstract: The antinociceptive action of three classes of gamma-aminobutyric acid (GABA) agonists was examined in mice using the hot-plate and tail-immersion tests. A significant increase in reaction time was noted in the hot-plate test after treatment with the direct-acting GABA receptor agonists, kojic amine or 4,5,6,7-tetrahydroisoxazolo[5,4-C] pyridin-3-ol, with gamma-vinyl GABA, an inhibitor of GABA degradation, or with nipecotic acid ethyl ester, an inhibitor of high-affinity GABA transport. Studies with naloxone indicated that the increase in pain threshold was not mediated through the brain opiate system, although it was possible to reverse the antinociceptive effect of these drugs with atropine. Receptor binding experiments indicated that except for the ethyl ester of nipecotic acid, the GABA agonists have little affinity for the cholinergic muscarinic receptor site. Atropine, at a dose that completely blocked the antinociceptive action of kojic amine, was unable to attenuate the sedative effects of this drug. These findings suggest that, regardless of their mechanism, the three types of GABA agonists tested are capable of inducing an antinociceptive response in mice and that this action is apparently secondary to a GABA-mediated increase in brain cholinergic function.
Abstract: Receptor binding studies were undertaken in an attempt to identify and characterize pharmacologically and functionally distinct receptor sites for gamma-aminobutyric acid (GABA) in rat brain. The results indicated that the potency of bicuculline, a GABA receptor antagonist, to displace membrane-bound [3H]GABA varies significantly among different brain regions, with the greatest potency found in the cerebral cortex and midbrain. In addition, in the presence of 50 mM ammonium thiocyanate, the potency of bicuculline to displace specifically bound [3H]GABA was increased significantly, with the magnitude of this increase being greater in some brain areas than others. The biological relevance of this thiocyanate-induced shift in the potency of bicuculline to inhibit [3H]GABA binding was indicated by the finding that ammonium thiocyanate also increased the potency of bicuculline to inhibit GABA-activated benzodiazepine receptor binding, a biochemical measure of GABA receptor function. Receptor site saturation analysis revealed that ammonium thiocyanate selectively abolished the high affinity GABA binding site without affecting either the low affinity component or GABA-activated benzodiazepine receptor binding. These findings provide further evidence for the existence of pharmacologically distinct GABA receptor sites, with some being more sensitive to the blocking action of bicuculline than others. Furthermore, the data provide direct evidence to support the hypothesis that only low affinity GABA receptor sites are linked to the benzodiazepine receptor, indicating that the kinetically different GABA binding sites are also functionally distinct. The discovery that ammonium thiocyanate selectively destroys high affinity GABA receptor binding may be useful for further defining the pharmacological, biochemical, and functional differences between GABA receptors in brain.
