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L N Johnson

Publications and source records attributed to L N Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1995

Crystal structure of the cell cycle-regulatory protein suc1 reveals a beta-hinge conformational switch.

The Schizosaccharomyces pombe cell cycle-regulatory protein suc1, named as the suppressor of cdc2 temperature-sensitive mutations, is essential for cell cycle progression. To understand suc1 structure-function relationships and to help resolve conflicting interpretations of suc1 function based on genetic studies of suc1 and its functional homologs in both lower and higher eukaryotes, we have determined the crystal structure of the beta-interchanged suc1 dimer. Each domain consists of three alpha-helices and a four-stranded beta-sheet, completed by the interchange of terminal beta-strands between the two subunits. This beta-interchanged suc1 dimer, when compared with the beta-hairpin single-domain folds of suc1, reveals a beta-hinge motif formed by the conserved amino acid sequence HVPEPH. This beta-hinge mediates the subunit conformation and assembly of suc1: closing produces the intrasubunit beta-hairpin and single-domain fold, whereas opening leads to the intersubunit beta-strand interchange and interlocked dimer assembly reported here. This conformational switch markedly changes the surface accessibility of sequence-conserved residues available for recognition of cyclin-dependent kinase, suggesting a structural mechanism for beta-hinge-mediated regulation of suc1 biological function. Thus, suc1 belongs to the family of domain-swapping proteins, consisting of intertwined and dimeric protein structures in which the dual assembly modes regulate their function.

Amino Acid Sequence↗
PubMed1995

Sulphate-activated phosphorylase b: the pH-dependence of catalytic activity.

The pH-dependence of sulphate-activated phosphorylase b has been studied in the direction of glycogen synthesis. The bell-shaped curve of the pH-dependence of the catalytic constant for the AMP-activated enzyme showed pK values of 6.1 and 7.3, but the curve for the enzyme activated by 0.9 M ammonium sulphate showed a drop of activity on the acid side at much higher pH values. Its bell was centred at pH 7.8 but it was too narrow to be characterized by only two pK values. The narrowness of the curve could be explained by positive co-operativity, but not its unusually steep acid side. We suggest that the fall on the acid side is due to more than one hydronation (addition of H+). The points can be fitted by a curve with two de-activating hydronations and a de-activating dehydronation having identical titration pK values of 7.5, and hence molecular values of 7.0, 7.5 and 8.0. If both 0.9 M ammonium sulphate and 5 mM AMP are added, the bell is as broad as with AMP alone, but is somewhat raised in pH optimum. The results are discussed in the light of new structural data from crystallographic studies on binary complexes of the enzyme.

Adenosine Monophosphate↗
PubMed1995

Glucose analogue inhibitors of glycogen phosphorylase: from crystallographic analysis to drug prediction using GRID force-field and GOLPE variable selection.

Several inhibitors of the large regulatory enzyme glycogen phosphorylase (GP) have been studied in crystallographic and kinetic experiments. GP catalyses the first step in the phosphorylysis of glycogen to glucose-l-phosphate, which is utilized via glycolysis to provide energy to sustain muscle contraction and in the liver is converted to glucose. alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase form b (GPb, K(i) = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several N-acetyl glucopyranosylamine derivatives have been synthesized and tested in a series of crystallographic and kinetic binding studies with GPb. The structural results of the bound enzyme-ligand complexes have been analysed together with the resulting affinities in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. Comparison of the N-methylacetyl glucopyranosylamine (N-methylamide, K(i) = 0.032 mM) with the analogous beta-methylamide derivative (C-methylamide, K(i) = 0.16 mM) illustrate the importance of forming good hydrogen bonds and obtaining complementarity of van der Waals interactions. These studies also have shown that the binding modes can be unpredictable but may be rationalized with the benefit of structural data and that a buried and mixed polar/non-polar catalytic site poses problems for the systematic addition of functional groups. Together with previous studies of glucose analogue inhibitors of GPb, this work forms the basis of a training set suitable for three-dimensional quantitative structure-activity relationship studies. The molecules in the training set are void of problems and potential errors arising from the alignment and bound conformations of each of the ligands since the coordinates were those determined experimentally from the X-ray crystallographic refined ligand-enzyme complexes. The computational procedure described in this work involves the use of the program GRID to describe the molecular structures and the progam GOLPE to obtain the partial least squares regression model with the highest prediction ability. The GRID/GOLPE procedure performed using 51 glucose analogue inhibitors of GPb has good overall predictivity [standard deviation of error predictions (SDEP) = 0.98 and Q(2) = 0.76] and has shown good agreement with the crystallographic and kinetic results by reliably selecting regions that are known to affect the binding affinity.

Journal Article↗
PubMed1995

Two structures of the catalytic domain of phosphorylase kinase: an active protein kinase complexed with substrate analogue and product.

BACKGROUND: Control of intracellular events by protein phosphorylation is promoted by specific protein kinases. All the known protein kinase possess a common structure that defines a catalytically competent entity termed the 'kinase catalytic core'. Within this common structural framework each kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Structural studies of phosphorylase kinase (Phk), the major substrate of which is glycogen phosphorylase, may be expected to shed light on its regulation. RESULTS: We report two crystal structures of the catalytic core (residues 1-298; Phk gamma trnc) of the gamma-subunit of rabbit muscle phosphorylase kinase: the binary complex with Mn2+/beta-gamma-imidoadenosine 5'-triphosphate (AMPPNP) to a resolution of 2.6 A and the binary complex with Mg2+/ADP to a resolution of 3.0 A. The structures were solved by molecular replacement using the cAMP-dependent protein kinase (cAPK) as a model. CONCLUSIONS: The overall structure of Phk gamma trnc is similar to that of the catalytic core of other protein kinases. It consists of two domians joined on one edge by a 'hinge', with the catalytic site located in the cleft between the domains. Phk gamma trnc is constitutively active, and lacks the need for an activatory phosphorylation event that is essential for many kinases. The structure exhibits an essentially 'closed' conformation of the domains which is similar to that of cAPK complexed with substrates. The phosphorylated residue that is located at the domain interface in many protein kinases and that is believed to stabilize an active conformation is substituted by a glutamate in Phk gamma trnc. The glutamate, in a similar manner to the phosphorylated residue in other protein kinases, interacts with an arginine adjacent to the catalytic aspartate but does not participate in interdomain contacts. The interactions between the enzyme and the nucleotide product of its activity, Mg2+/ADP, explain the inhibitory properties of the nucleotides that are observed in kinetic studies.

Adenosine Diphosphate↗
PubMed1995

Effects of novel analogues of D-glucose on glycogen phosphorylase activities in crude extracts of liver and skeletal muscle.

The inhibitory properties of a series of both N-linked and C-linked C1-substituted glucose derivatives towards glycogen phosphorylase (GP) activity from crude extracts of rat liver and muscle have been measured. The most effective inhibitor was N-acetyl-beta-D-glucopyranosylamine, which has Kis of 51 microM (muscle GPa), 30 microM (muscle GPb), 2.7 mM (liver GPa) and 4 mM (liver GPb). All analogues tested inhibit muscle GP more potently than liver GP, highlighting some differences between the two isoenzymes, which are nearly 80% similar. The human liver GP enzyme has been modelled on the basis of the rabbit muscle structure and, together with comparison of structures of muscle GPa and GPb, has provided some insights into possible explanations for the different properties of the two isoenzymes. Maximal activities of GP have also been measured in tissues from diabetic (db/db) and wild-type (db/+) mice. Liver GP from db/db mice exhibits higher activity [132% (a)-67% (b)] than from db/+ controls, although similar activities were observed for muscle GP from both db/db and db/+ animals.

Amino Acid Sequence↗
PubMed1995

The crystal structure of p13suc1, a p34cdc2-interacting cell cycle control protein.

p13suc1 binds to p34cdc2 kinase and is essential for cell cycle progression in eukaryotic cells. The crystal structure of S.pombe p13suc1 has been solved to 2.7 A resolution using data collected at the ESRF source, Grenoble, from both native crystals and crystals of a seleno-methionine derivative. The starting point for structure solution was the determination of the six selenium sites by direct methods. The structure is dominated by a four-stranded beta-sheet, with four further alpha-helical regions. p13suc1 crystallizes as a dimer in the asymmetric unit stabilized by the binding of two zinc ions. A third zinc site stabilizes the higher-order crystal packing. The sites are consistent with a requirement for zinc during crystal growth. A likely site for p13suc1-protein interaction is immediately evident on one face of the p13suc1 surface. This region comprises a group of conserved, exposed aromatic and hydrophobic residues below a flexible negatively charged loop. A conserved positively charged area would also present a notable surface feature in the monomer, but is buried at the dimer interface. p13suc1 is larger than its recently solved human homologue p9CKS2, with the extra polypeptide forming a helical N-terminal extension and a surface loop between alpha-helices 3 and 4. Notably, p13suc1 does not show the unusual beta-strand exchange that creates an intimate p9CKS2 dimer. p13suc1 cannot oligomerize to form a stable hexamer as has been proposed for p9CKS2.

Amino Acid Sequence↗
PubMed1995

Expression, purification and crystallisation of phosphorylase kinase catalytic domain.

The catalytic subunit of phosphorylase kinase is composed of a kinase catalytic core domain (residues 1 to 298), which has a 33% identity with the kinase core of the cyclic AMP-dependent protein kinase, and a C-terminal calmodulin binding domain. The kinase domain of the catalytic subunit has been expressed in Escherichia coli, purified and crystallised in the presence of ATP and magnesium from 5% (w/v) polyethylene glycol 8000, 10% (v/v) glycerol, 50 mM Hepes/NaOH (pH 6.9). A three-fold excess of magnesium to ATP was used for crystal growth. The inclusion of glycerol in the crystallization medium produced a marked reduction in mosaic spread of the diffraction spots from greater than 1 degree to 0.3 degree. The crystals are orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions a = 47.1 A, b = 69.1 A, c = 112.9 A and one molecule per asymmetric unit. Data to 3 A resolution have been collected and structure determination is in progress.

Animals↗
PubMed1995

N-acetyl-beta-D-glucopyranosylamine: a potent T-state inhibitor of glycogen phosphorylase. A comparison with alpha-D-glucose.

Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to alpha-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of beta-D-glucopyranosylamine, N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (Ki = 32 microM) and the a (Ki = 35 microM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase a and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 A resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of alpha-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-alpha-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.

Animals↗
PubMed1995

Confrontation visual field loss as a function of decibel sensitivity loss on automated static perimetry. Implications on the accuracy of confrontation visual field testing.

PURPOSE: To evaluate the accuracy of confrontation visual field testing with regard to the density of the visual field defect and its location in the peripheral visual field. METHODS: A prospective comparison of confrontation visual field testing with full-threshold Humphrey automated static perimetry C24-2 or C30-2 was conducted at a university eye center over a 3-month period. Seventy-two patients with a variety of neurologic and ophthalmologic conditions underwent confrontation visual field testing and automated perimetry as a part of their evaluation. One visual field from each patient was analyzed for this study. RESULTS: Confrontation visual field testing yielded an overall sensitivity for detecting an abnormal visual field (full-field analysis) of 63%, when sensitivity of confrontation testing rested on the detection of just one abnormal quadrant. The sensitivity of confrontation testing varied depending on the type of visual field loss present: 51% for arcuate scotomas, 67% for visual field constriction, 78% for altitudinal scotomas, and 90% for hemianopias. The sensitivity of detecting abnormal visual field quadrants, rather than the full-field analysis, was, however, poor at 38%. The sensitivity of confrontation testing was lower for superior quadrant defects and higher for inferior quadrant defects. The estimated probability of detecting an abnormal visual field quadrant occurring at a -26-decibel sensitivity loss from age-matched healthy patients for superior quadrant defects and a -19-decibel sensitivity loss for inferior quadrant defects was 50%. The increased sensitivity noted for visual field defects and for inferior quadrant defects appears to be related, in part, to the density of the visual field loss present. CONCLUSION: Confrontation visual field testing is relatively insensitive unless a moderate to dense defect is present, and as such is a poor screening test. However, when visual field defects are identified with confrontation visual field testing, the defects often are real as per the high specificity (97%) and high positive predictive value (96%).

Adult↗
PubMed1994

Crystal structure of the mutant D52S hen egg white lysozyme with an oligosaccharide product.

The crystal structure of a mutant hen egg white lysozyme, in which the key catalytic residue aspartic acid 52 has been changed to a serine residue (D52S HEWL), has been determined and refined to a crystallographic R value of 0.173 for all data F > 0 between 8 and 1.9 A resolution. The D52S HEWL structure is very similar to the native HEWL structure (r.m.s. deviation of main-chain atoms 0.20 A). Small shifts that result from the change in hydrogen bonding pattern on substitution of Asp by Ser were observed in the loop between beta-strands in the region of residues 46 to 49. D52S HEWL exhibits less than 1% activity against the bacterial cell wall substrate. Cocrystallisation experiments with the hexasaccharide substrate beta(1-4) polymer of N-acetyl-D-glucosamine (GlcNAc6) resulted in crystals between 5 days and 14 days after the initial mixing of enzyme and substrate. Analysis by laser absorption mass spectrometry of the oligosaccharides present after incubation with native and D52S HEWL under conditions similar to those used for crystal growth showed that after 14 days with native HEWL complete catalysis to GlcNAc3. GlcNAc2 and GlcNac had occurred but with D52S HEWL only partial catalysis to the major products GlcNAc4 and GlcNAc2 had occurred and at least 50% of the GlcNAc6 remained intact. X-ray analysis of the D52S-oligosaccharide complex crystals showed that they contained the product GlcNAc4. The structure of the D52S HEWL-GlcNAc4 complex has been determined and refined to an R value of 0.160 for data between 8 and 2 A resolution. GlcNAc4 occupies sites A to D in the active site cleft. Careful refinement and examination of 2Fo-Fc electron density maps showed that the sugar in site D has the sofa conformation, a conformation previously observed with the HEWL complex with tetra-N-acetylglucosamine lactone transition state analogue, the HEWL complex with the cell wall trisaccharide and the phage T4 lysozyme complex with a cell wall product. The semi-axial C(5)-C(6) geometry of the sofa is stabilised by hydrogen bonds from the O-6 hydroxyl group to the main-chain N of Val109 and main-chain O of Ala107. The sugar in site D adopts the alpha configuration, seemingly in conflict with the observation that the hydrolysis of beta (1-4) glycosidie linkage by HEWL proceeds with 99.9% retention of beta-configuration.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Design of inhibitors of glycogen phosphorylase: a study of alpha- and beta-C-glucosides and 1-thio-beta-D-glucose compounds.

alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase b (Ki = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that, in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several alpha- and beta-anhydroglucoheptonic acid derivatives and 1-deoxy-1-thio-beta-D-glucose analogues have been synthesized and tested in a series of crystallographic and kinetic binding studies with glycogen phosphorylase. The structural results of the bound enzyme-ligand complexes have been analyzed, together with the resulting affinities, in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. This work has shown the following: (i) Similar affinities may be obtained through different sets of interactions. Specifically, in the case of the alpha- and beta-glucose-C-amides, similar Ki's (0.37 and 0.44 mM, respectively) are obtained with the alpha-anomer through interactions from the ligand via water molecules to the protein and with the beta-anomer through direct interaction from the ligand to the protein. Thus, hydrogen bonds through water can contribute binding energy similar to that of hydrogen bonds directly to the protein. (ii) Attempts to improve the inhibition by additional groups did not always lead to the expected result. The addition of nonpolar groups to the alpha-carboxamide resulted in a change in conformation of the pyranose ring from a chair to a skew boat and the consequent loss of favorable hydrogen bonds and increase in the Ki. (iii) The addition of polar groups to the alpha-carboxamide led to compounds with the chair conformation, and in the examples studied, it appears that hydration by a water molecule may provide sufficient stabilization to retain the chair conformation. (iv) The best inhibitor was N-methyl-beta-glucose-C-carboxamide (Ki = 0.16 mM), which showed a 46-fold improvement in Ki from the parent beta-D-glucose. The decrease in Ki may be accounted for by a single hydrogen bond from the amide nitrogen to a main-chain carbonyl oxygen, an increase in entropy through displacement of a water molecule, and favorable van der Waals contacts between the methyl substituent and nonpolar protein residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Laue and monochromatic diffraction studies on catalysis in phosphorylase b crystals.

The conversion of substrate, heptenitol, to product, beta-1-C-methyl, alpha-D-glucose-1-phosphate (heptulose-2-P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5-dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a xenon flash lamp, was monitored in the crystal with a diode array spectrophotometer. In the Laue diffraction experiments, data to 2.8 A resolution were collected and the first time shot was obtained at 3 min from the start of reaction, and data collection comprised three 800-ms exposures. Careful data processing of Laue photographs for the large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference maps obtained from the Laue measurements showed that very little catalysis had occurred 3 min and 1 h after release of phosphate, and a distinct peak consistent with the position expected for phosphate, in the attacking position was observed. Data collection times with monochromatic crystallographic methods on a home source took 16 h for data to 2.3 A resolution. Sufficient phosphate was released from the caged phosphate in the crystal from 5 flashes with a xenon flashlamp within 1 min for the reaction to go to completion within the time scale of the monochromatic data collection procedures. The heptulose-2-P product complex has been refined and the model agrees with that obtained previously with the major difference that the interchange of an aspartic acid (Asp 283) by an arginine (Arg 569) was not observed at the catalytic site. This change is part of the activation process of glycogen phosphorylase and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conformational change, and because activators of the enzymic reaction were not present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer photons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through crystallographic refinement at 2.3 A resolution to establish the fractional occupancies of the enzyme-substrate and enzyme-product complexes, but the results did indicate that the reaction was proceeding slowly, consistent with approximate calculations for the likely rate of the reaction in the crystal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

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↗
PubMed1994

Mutational analysis supports a structural model for the cell cycle protein kinase p34.

Structural models for the eukaryotic cell cycle control protein p34 from human, S. pombe and S. cerevisiae have been derived from the crystallographic coordinates of the cAMP-dependent protein kinase (cAPK) catalytic subunit (active conformation) and compared with the structure of inactive CDK2 apoenzyme. Differences between the p34 and cAPK catalytic sites provide a possible explanation for their different substrate specificities. The p34 models localize Tyr15 and Thr14 close to the sites of catalysis and substrate recognition where their phosphorylation could inhibit p34 kinase activity either by blocking MgATP or substrate binding. The conserved sequences PSTAIRE and LYLIFEFL are both close to the catalytic site and accessible on the protein surface available to mediate interactions with other proteins. It is predicted that p34 has an active-site cleft composed almost entirely of sequences common to all protein kinases and sequences unique to the p34 protein family. Genetic and biochemical analyses of p34 have shown that it interacts extensively with a number of other proteins. The model allows the relative disposition of these sites of mutation to each other and to the sites of catalysis and substrate recognition to be appreciated. Surface regions on p34 that are important for function have been identified. These sites identify residues that may interact with p13suc1, cyclin, p107wee1 and p80cdc25.

Amino Acid Sequence↗