Biomedical subjects
L N Johnson
Publications and source records attributed to L N Johnson.
The crystal structure of the Escherichia coli maltodextrin phosphorylase-acarbose complex.
Acarbose is a naturally occurring pseudo-tetrasaccharide. It has been used in conjunction with other drugs in the treatment of diabetes where it acts as an inhibitor of intestinal glucosidases. To probe the interactions of acarbose with other carbohydrate recognition enzymes, the crystal structure of E. coli maltodextrin phosphorylase (MalP) complexed with acarbose has been determined at 2.95 A resolution and refined to crystallographic R-values of R (Rfree) = 0.241 (0.293), respectively. Acarbose adopts a conformation that is close to its major minimum free energy conformation in the MalP-acarbose structure. The acarviosine moiety of acarbose occupies sub-sites +1 and +2 and the disaccharide sub-sites +3 and +4. (The site of phosphorolysis is between sub-sites -1 and +1.) This is the first identification of sub-sites +3 and +4 of MalP. Interactions of the glucosyl residues in sub-sites +2 and +4 are dominated by carbohydrate stacking interactions with tyrosine residues. These tyrosines (Tyr280 and Tyr613, respectively, in the rabbit muscle phosphorylase numbering scheme) are conserved in all species of phosphorylase. A glycerol molecule from the cryoprotectant occupies sub-site -1. The identification of four oligosaccharide sub-sites, that extend from the interior of the phosphorylase close to the catalytic site to the exterior surface of MalP, provides a structural rationalization of the substrate selectivity of MalP for a pentasaccharide substrate. Crystallographic binding studies of acarbose with amylases, glucoamylases, and glycosyltranferases and NMR studies of acarbose in solution have shown that acarbose can adopt two different conformations. This flexibility allows acarbose to target a number of different enzymes. The two alternative conformations of acarbose when bound to different carbohydrate enzymes are discussed.
Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity.
We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-A resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.
David Phillips and the origin of structural enzymology.
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The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases.
Progression through the eukaryotic cell cycle is driven by the orderly activation of cyclin-dependent kinases (CDKs). For activity, CDKs require association with a cyclin and phosphorylation by a separate protein kinase at a conserved threonine residue (T160 in CDK2). Here we present the structure of a complex consisting of phosphorylated CDK2 and cyclin A together with an optimal peptide substrate, HHASPRK. This structure provides an explanation for the specificity of CDK2 towards the proline that follows the phosphorylatable serine of the substrate peptide, and the requirement for the basic residue in the P+3 position of the substrate. We also present the structure of phosphorylated CDK2 plus cyclin A3 in complex with residues 658-668 from the CDK2 substrate p107. These residues include the RXL motif required to target p107 to cyclins. This structure explains the specificity of the RXL motif for cyclins.
Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure.
The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.
The basic skills evaluation device.
The Commission on Accreditation for Marriage and Family Therapy Education (COAMFTE) requires that accredited programs evaluate trainees' clinical skills at various times during trainees' programs. The Commission does not attempt, however, to describe for programs either the nature of the skills nor how they are to be evaluated, leaving this to the programs to create. This paper describes the development and uses of the Basic Skills Evaluation Device (BSED). Using a published list of skills (Figley & Nelson, 1989), data from the literature, and data collected from COAMFTE accredited and candidacy programs, the authors and their colleagues developed a device for supervisors to use in evaluating beginning family therapists. Data regarding the reliability of the device are presented, along with limitations and suggestions for various ways of using the device.
Toxic optic neuropathy after concomitant use of melatonin, zoloft, and a high-protein diet.
Melatonin is a neuromodulating hormone found in the pineal gland and retina. It is involved in light-dark circadian rhythms and mediates retinal processes in a manner antagonistic to that of dopamine. Zoloft (sertraline) is an antidepressant drug that blocks the reuptake of serotonin at the neural synapse. Serotonin is the natural precursor of melatonin. A 42-year-old woman sought treatment for visual acuity loss, dyschromatopsia, and altered light adaptation. Neuro-ophthalmologic examination was otherwise normal except for evolving bilateral cecocentral scotomas. She had taken Zoloft for 4 years and began a high-protein diet with melatonin supplementation 2 weeks before onset of visual symptoms. Visual acuity and color vision improved within 2 months after melatonin and the high-protein diet were discontinued. Combined use of melatonin, Zoloft, and a high-protein diet may have resulted in melatonin/dopamine imbalance in the retina, manifesting as a toxic optic neuropathy. Physicians and patients should be alerted to this potential drug interaction.
The Eleventh Datta Lecture. The structural basis for substrate recognition and control by protein kinases.
Protein kinases catalyse phospho transfer reactions from ATP to serine, threonine or tyrosine residues in target substrates and provide key mechanisms for control of cellular signalling processes. The crystal structures of 12 protein kinases are now known. These include structures of kinases in the active state in ternary complexes with ATP (or analogues) and inhibitor or peptide substrates (e.g. cyclic AMP dependent protein kinase, phosphorylase kinase and insulin receptor tyrosine kinase); kinases in both active and inactive states (e.g. CDK2/cyclin A, insulin receptor tyrosine kinase and MAPK); kinases in the active state (e.g. casein kinase 1, Lck); and kinases in inactive states (e.g. twitchin kinase, calcium calmodulin kinase 1, FGF receptor kinase, c-Src and Hck). This paper summarises the detailed information obtained with active phosphorylase kinase ternary complex and reviews the results with reference to other kinase structures for insights into mechanisms for substrate recognition and control.
The structure of a glycogen phosphorylase glucopyranose spirohydantoin complex at 1.8 A resolution and 100 K: the role of the water structure and its contribution to binding.
A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (R[free] 0.248) and 0.182 (R[free] 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ((B) room temperature GPb = 34.9 A2; (B) 100 K GPb = 23.4 A2). The glucopyranose spirohydantoin inhibitor (Ki = 3.0 microM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10(3), makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.
The role of weight loss and acetazolamide in the treatment of idiopathic intracranial hypertension (pseudotumor cerebri)
OBJECTIVE: To determine the weight loss associated with resolution of papilledema from idiopathic intracranial hypertension (IIH). DESIGN: A retrospective study. PARTICIPANTS: Fifteen consecutive female patients with IIH associated with obesity were studied. INTERVENTION: Patients underwent weight loss and treatment with acetazolamide during a 24-week period. MAIN OUTCOME MEASURES: The severity of papilledema was graded: absent (grade 0), mild (grade 1), moderate (grade 2), and marked (grade 3), based on a predetermined grading system ("gold standard") using stereoscopic photographs and the Frisén classification. RESULTS: The 15 patients, with mean age of 31.3+/-8.8 years, had a mean weight of 110.5+/-28.7 kg and mean body mass index of 40.7+/-13.0 kg/m2. Eleven (73.3%) patients had improved papilledema during the 24-week study period, of which 10 (66.7%) had complete resolution of papilledema within a median time of 8.5 weeks. An average of 3.3% weight loss (+/-0.5% standard error of the mean) was observed among patients having a one-grade change in papilledema. Weight loss of 6.2%+/-0.6% standard error of the mean was associated with a three-grade change in papilledema (i.e., complete resolution of marked papilledema). Nine of the ten patients with complete resolution of papilledema also took acetazolamide. However, none (26.7%) of the four patients without weight loss had improvement in papilledema despite similar treatment with acetazolamide. CONCLUSIONS: Approximately 6% weight loss was associated with resolution of marked papilledema in these authors' patients. The benefit of acetazolamide in IIH is questioned since weight loss, rather than acetazolamide, appeared to have been the catalyst for reducing the severity of papilledema.
The early history of lysozyme.
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The 5-year risk of MS after optic neuritis.
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Nonarteritic anterior ischemic optic neuropathy.
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The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition.
The structure of a truncated form of the gamma-subunit of phosphorylase kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered.
The structure of glycogen phosphorylase b with an alkyldihydropyridine-dicarboxylic acid compound, a novel and potent inhibitor.
BACKGROUND: In muscle and liver, glycogen concentrations are regulated by the reciprocal activities of glycogen phosphorylase (GP) and glycogen synthase. An alkyl-dihydropyridine-dicarboxylic acid has been found to be a potent inhibitor of GP, and as such has potential to contribute to the regulation of glycogen metabolism in the non-insulin-dependent diabetes diseased state. The inhibitor has no structural similarity to the natural regulators of GP. We have carried out structural studies in order to elucidate the mechanism of inhibition. RESULTS: Kinetic studies with rabbit muscle glycogen phosphorylase b (GPb) show that the compound (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5, 6-tricarboxylate (Bay W1807) has a Ki = 1.6 nM and is a competitive inhibitor with respect to AMP. The structure of the cocrystallised GPb-W1807 complex has been determined at 100K to 2.3 A resolution and refined to an R factor of 0.198 (Rfree = 0.287). W1807 binds at the GPb allosteric effector site, the site which binds AMP, glucose-6-phosphate and a number of other phosphorylated ligands, and induces conformational changes that are characteristic of those observed with the naturally occurring allosteric inhibitor, glucose-6-phosphate. The dihydropyridine-5,6-dicarboxylate groups mimic the phosphate group of ligands that bind to the allosteric site and contact three arginine residues. CONCLUSIONS: The high affinity of W1807 for GP appears to arise from the numerous nonpolar interactions made between the ligand and the protein. Its potency as an inhibitor results from the induced conformational changes that lock the enzyme in a conformation known as the T' state. Allosteric enzymes, such as GP, offer a new strategy for structure-based drug design in which the allosteric site can be exploited. The results reported here may have important implications in the design of new therapeutic compounds.
The crystal structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase.
In animals, glycogen phosphorylase (GP) exists in an inactive (T state) and an active (R state) equilibrium that can be altered by allosteric effectors or covalent modification. In Escherichia coli, the activity of maltodextrin phosphorylase (MalP) is controlled by induction at the level of gene expression, and the enzyme exhibits no regulatory properties. We report the crystal structure of E. coli maltodextrin phosphorylase refined to 2.4 A resolution. The molecule consists of a dimer with 796 amino acids per monomer, with 46% sequence identity to the mammalian enzyme. The overall structure of MalP shows a similar fold to GP and the catalytic sites are highly conserved. However, the relative orientation of the two subunits in E. coli MalP is different from both the T and R state GP structures, and there are significant changes at the subunit-subunit interfaces. The sequence changes result in loss of each of the control sites present in rabbit muscle GP. As a result of the changes at the subunit interface, the 280s loop, which in T state GP acts as a gate to control access to the catalytic site, is held in an open conformation in MalP. The open access to the conserved catalytic site provides an explanation for the activity without control in this basic archetype of a phosphorylase.
The cyclin box fold: protein recognition in cell-cycle and transcription control.
Regulation of both the cell cycle and gene transcription is essential for orderly progression of cell growth and division. Recent results on the structures of two cyclins, cyclin A and cyclin H, and two transcription factor mediator proteins, TFIIB and the A pocket region of the retinoblastoma tumour suppressor protein (Rb), show that they share domains with a strikingly similar alpha-helical topology, despite remote sequence identity.