Hijacking of a substrate-binding protein scaffold for use in mycobacterial cell wall biosynthesis

The waxy cell wall is crucial to the survival of mycobacteria within the infected host. The cell wall is a complex structure rich in unusual molecules that includes two related lipoglycans, the phosphatidylinositol mannosides (PIMs) and lipoarabinomannans (LAMs). Many proteins implicated in the PIM/LAM biosynthetic pathway, while attractive therapeutic targets, are poorly defined. The 2.4A resolution crystal structure of an essential lipoprotein, LpqW, implicated in LAM biosynthesis is reported here. LpqW adopts a scaffold reminiscent of the distantly related, promiscuous substrate-binding proteins of the ATP-binding cassette import system. Nevertheless, the unique closed conformation of LpqW suggests that mycobacteria and other closely related pathogens have hijacked this scaffold for use in key processes of cell wall biosynthesis. In silico docking provided a plausible model in which the candidate PIM ligand binds within a marked electronegative region located on the surface of LpqW. We suggest that LpqW represents an archetypal lipoprotein that channels intermediates from a pathway for mature PIM production into a pathway for LAM biosynthesis, thus controlling the relative abundance of these two important components of the cell wall.     

Substrate requirements and subcellular distribution of calcium transport activities in brain membranes

Ca²⁺ transport activity in synaptosomal membranes has been identified as having two major components: Ca²⁺-stimulated ATP hydrolysis and ATP-dependent CA²⁺ uptake. Both processes exhibit similar affinities for Ca²⁺ and operate maximally under identical buffer conditions. Subcellular fractionation studies revealed the Ca²⁺/Mg²⁺ ATPase and ATP-dependent CA²⁺ uptake activities to be highest in synaptic plasma membrane fractions 1 and 2, with lesser activity in synaptic vesicles and mitochondria. Progressive treatment with Triton X-100 activated, then decreased Ca²⁺/Mg²⁺ ATPase, Mg²⁺ ATPase and Ca²⁺ ATPase. ATP-dependent Ca²⁺ uptake was progressively decreased by similar treatment with Triton X-100. These studies illustrate that Ca²⁺ ATPase and ATP-dependent Ca²⁺ uptake may provide two important mechanisms for buffering of cytosolic Ca²⁺ at the nerve terminal. These systems may function to rapidly sequester cytosolic Ca²⁺ following a rise during depolarization and then extrude Ca²⁺ from the terminal against a concentration gradient. This regulation of cytosolic Ca²⁺, represented by two processes of the type seen in other plasma membranes, may play critical roles in calcium homeostasis in nerve cells.Footnote: Portions of this research were submitted by K. M. Garrett in partial fulfillment of requirements for the Doctor of Philosophy Degree in Pharmacology at the University of Texas Health Science Center.     

Modern antipsychotic drugs: a critical overview

CONVENTIONAL ANTIPSYCHOTIC DRUGS, used for a half century to treat a range of major psychiatric disorders, are being replaced in clinical practice by modern “atypical” antipsychotics, including aripiprazole, clozapine, olanzapine, quetiapine, risperidone and ziprasidone among others. As a class, the newer drugs have been promoted as being broadly clinically superior, but the evidence for this is problematic. In this brief critical overview, we consider the pharmacology, therapeutic effectiveness, tolerability, adverse effects and costs of individual modern agents versus older antipsychotic drugs. Because of typically minor differences between agents in clinical effectiveness and tolerability, and because of growing concerns about potential adverse long-term health consequences of some modern agents, it is reasonable to consider both older and newer drugs for clinical use, and it is important to inform patients of relative benefits, risks and costs of specific choices.Antipsychotic drugs are useful for treating a range of severe psychiatric disorders. Applications include the short-term treatment of acute psychotic, manic and psychotic-depressive disorders as well as agitated states in delirium and dementia and the long-term treatment of chronic psychotic disorders including schizophrenia, schizoaffective disorder and delusional disorders. Newer, “second-generation” antipsychotic drugs have largely replaced older phenothiazine, thioxanthene and butyrophenone neuroleptics in clinical practice (1^,2 The development of modern antipsychotic drugs was stimulated by a landmark 1988 study that showed clozapine to be superior in efficacy to chlorpromazine in schizophrenia patients resistant to high doses of haloperidol and to have none of the adverse neurologic effects typical of older antipsychotic agents.³ Clozapine was considered “atypical” in having a very low risk of adverse extrapyramidal symptoms. This term has since been applied broadly and uncritically to antipsychotic drugs marketed in the past decade, despite their striking chemical, pharmacologic and clinical heterogeneity.⁴ In this overview we consider the neuropharmacology, efficacy and adverse effects of conventional antipsychotics and specific modern antipsychotic drugs.Table 1     

Pressure dissociation of integration host factor-DNA complexes reveals flexibility-dependent structural variation at the protein-DNA interface

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236–39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379–401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF–DNA interfaces while the resulting energetic compensation maintains the same net binding energy.     

Membrane association of the PTEN tumor suppressor: molecular details of the protein-membrane complex from SPR binding studies and neutron reflection

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P₂) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P₂ were determined to be K_d∼12 µM and 0.4 µM, respectively, and K_d∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P₂. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P₂ and an increased apparent affinity to PI(3,4,5)P₃, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P₂ and no synergy in its binding with PS and PI(4,5)P₂. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.     

Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides

A -galactosidase (EC 3.2.1.23) capable of degrading a number of fruit cell-wall polysaccharides in vitro, was isolated from ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson cv. Hayward). The enzyme has a molecular weight of approximately 60 kDa by gel permeation and consists of several basic isoforms. Several polypeptides were enriched during purification, with 33-, 46- and 67-kDa bands being predominant after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme against p-nitrophenyl--d-galactopyranoside was at pH 3.2, but against a galactan purified from kiwifruit cell walls, it was at pH 4.9. The enzyme was specific for galactosyl residues in the -configuration, releasing galactose from a variety of kiwifruit cell-wall polysaccharide fractions including cell wall material, Na₂CO₃-soluble pectin, high-molecular-weight galactan, xyloglucan, and galactoglucomannan. A galactosylated glucuronomannan found throughout the kiwifruit plant was also a substrate for the enzyme. The results indicate that the enzyme attacks the non-reducing end of galactose side chains, cleaving single galactose residues which may be attached to the 2, 3, 4, or 6 position of the aglycone. Activity of the enzyme in-vitro was too low to account for the total loss of galactose from the cell walls during ripening. If the -galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening then its in-vivo activity must be much greater than that observed in-vitro.Abbreviations CWM cell wall material - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Bronwyn Culling and Teresa Wegrzyn for assistance and acknowledge a contribution towards the cost of the research from the New Zealand Kiwifruit Marketing Board.     

The effect of intrinsic curvature on supercoiling: Predictions of elasticity theory

Elasticity theory of naturally curved rods is employed to study the effects of intrinsic curvature on the properties of the equilibrium conformations of supercoiled DNA. The results stand in sharp contrast to those obtained when the molecule is viewed as being straight in its relaxed form. Starting from very fundamental principles of the theory, we show that the torsion of an open segment with a curved duplex axis can vary when the temperature, and along with it, the intrinsic twist is changed. Conversely, an imposed helicity, such as might be associated with binding to a histone, can change the intrinsic twist. It is also shown that another consequence of the presence of naturally curved sequences is that the twist density will, in general, vary with position along the chain in all equilibrium states. Then portions of the molecule will be more or less susceptible to interaction with other agents sensitive to such a variation. Finally, some closed equilibrium global structures uniquely associated with intrinsic curvature are discussed. © 1993 John Wiley & Sons, Inc.     

The effects of carbon monoxide and hypoxic hypoxia on amine-containing cells in the tracheal epithelium of young rabbits

Summary Endogenously fluorescent, singly occurring, amine-containing cells in tracheal epithelium were examined in 3-, 10-, and 28-day-old rabbits. These cells are pyramidal in shape with the apex projected toward the tracheal lumen. The cytoplasm exhibits a yellow fluorescence which is predominantly supranuclear. Occasional, infranuclear, fluorescent cytoplasmic processes project from the cells. The numbers of fluorescent cells per unit length of trachea increase with age. Acute exposure of 10-day-old rabbits to 13% O₂ decreases the number of detectable fluorescent cells in the trachea compared to controls exposed to room air. Similarly, exposure to 750 ppm carbon monoxide decreases the number of fluorescent epithelial cells appearing in tracheas of 10- and 28-day-old rabbits. These results suggest that the amine-containing epithelial cells of the trachea respond to tissue hypoxia and that decreased airway pO₂ is not necessary to elicit a response.Supported by a grant from The Council for Tobacco Research, U.S.A., Inc. We are grateful to Margaret Hogan and Scott Pine for technical assistance     

Membrane receptors of mouse leukocytes. I. Two types of complement receptors for different regions of C3

Mouse leukocytes were studied for membrane receptors for the third C component by rosette formation with C coated erythrocytes (EAC). Methods were devised for the preparation of EAC complexes containing either mouse C3b or mouse C3d. EAC 1-3dmo were prepared from EA treated with whole mouse serum while EAC 1-3bmo were produced from EAC 142hu treated with whole mouse serum containing sodium suramin. The specificity of the EAC complexes for mouse leukocytes was confirmed by inhibition experiments using fluid phase human C3d. Low concentrations of fluid phase human C3d inhibited EAC1-3dmo rosettes but failed to inhibit EAC 1-3bmo rosettes. Eight-fold higher concentrations of fluid phase C3d caused partial inhibition of EAC1-3bmo rosette formation with lymphocytes, but not with other types of murine leukocytes. Thus mouse leukocytes apparently contain the same two types of C receptors as do human and guinea pig leukocytes. Mouse CR1 is specific for a non-C3d region of C3b, (possibly analogous to human C3c) whereas mouse CR2 is specific for both C3d and the C3d region of C3b.     

Diphtheria toxin-receptor interaction: A polyphosphate-insensitive diphtheria toxin-binding domain

Inositol hexaphosphate, and other polyphosphates, inhibit diphtheria toxin-mediated cytotoxicity by binding to the toxin at a highly cationic site called the P site and preventing toxin binding to cell surface receptors. The binding of diphtheria toxin to a solubilized cell surface glycoprotein (150,000 daltons) is also inhibited by these polyphosphates. Treatment of this 150,000 dalton diphtheria toxin-binding cell surface glycoprotein with papain yielded an 88,000 dalton and a 74,000 dalton diphtheria toxin-binding glycoprotein whose binding to toxin was no longer inhibited by inositol hexaphosphate. This result suggests a model of diphtheria toxin-receptor interaction in which the toxin receptor possesses one binding site which interacts with the P site of the toxin in a $\text{polyphosphate-sensitive}$ fashion, and another binding site (located within the papain-derived 74,000–88,000 dalton glycoproteins) which can interact with the toxin at a site distinct from the P site (the X site) in a $\text{polyphosphate-insensitive}$ fashion. This X site-receptor interaction may be involved in the binding of CRM proteins that bind to the toxin receptor but that do not bind polyphosphates, or it may be involved in the entry process of the toxin.