A protein residing at the subunit interface of the bacterial ribosome

Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.

Relative positioning of classical benzodiazepines to the γ2-subunit of GABAA receptors.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site at the α/γ subunit interface on some of these receptors. Diazepam has sedative, hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects. It acts by potentiating the current evoked by the agonist GABA. Understanding specific interaction of benzodiazepines in the binding pocket of different GABAA receptor isoforms might help to separate these divergent effects. As a first step, we characterized the interaction between diazepam and the major GABAA receptor isoform α1β2γ2. We mutated several amino acid residues on the γ2-subunit assumed to be located near or in the benzodiazepine binding pocket individually to cysteine and studied the interaction with three ligands that are modified with a cysteine-reactive isothiocyanate group (-NCS). When the reactive NCS group is in apposition to the cysteine residue this leads to a covalent reaction. In this way, three amino acid residues, γ2Tyr58, γ2Asn60, and γ2Val190 were located relative to classical benzodiazepines in their binding pocket on GABAA receptors.

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.

Conformational differences between the wild type and V30M mutant transthyretin modulate its binding to genistein: Implications to tetramer stability and ligand-binding

Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. It was recently found that the isoflavone genistein (GEN) potently inhibits TTR amyloid fibril formation (Green et al., 2005) and is therefore a promising candidate for TTR amyloidosis treatment. Here we used structural and biophysical approaches to characterize genistein binding to the wild type (TTRwt) and to its most frequent amyloidogenic variant, the V30M mutant. In a dose-dependent manner, genistein elicited considerable increases in both mutant and TTRwt stability as demonstrated by high hydrostatic pressure (HHP) and acid-mediated dissociation/denaturation assays. TTR:GEN crystal complexes and isothermal titration calorimetry (ITC) experiments showed that the binding mechanisms of genistein to the TTRwt and to V30M are different and are dependent on apoTTR structure conformations. Furthermore, we could also identify potential allosteric movements caused by genistein binding to the wild type TTR that explains, at least in part, the frequently observed negatively cooperative process between the two sites of TTRwt when binding ligands. These findings show that TTR mutants may present different ligand recognition and therefore are of value in ligand design for inhibiting TTR amyloidosis. (C) 2010 Elsevier Inc. All rights reserved.

A closer look at the high affinity benzodiazepine binding site on GABAA receptors

Ligands of the benzodiazepine binding site of the GABA(A) receptor come in three flavors: positive allosteric modulators, negative allosteric modulators and antagonists all of which can bind with high affinity. The GABA(A) receptor is a pentameric protein which forms a chloride selective ion channel and ligands of the benzodiazepine binding site stabilize three different conformations of this protein. Classical benzodiazepines exert a positive allosteric effect by increasing the apparent affinity of channel opening by the agonist γ-aminobutyric acid (GABA). We concentrate here on the major adult isoform, the α(1)β(2)γ(2) GABA(A) receptor. The classical binding pocket for benzodiazepines is located in a subunit cleft between α(1) and γ(2) subunits in a position homologous to the agonist binding site for GABA that is located between β(2) and α(1) subunits. We review here approaches to this picture. In particular, point mutations were performed in combination with subsequent analysis of the expressed mutant proteins using either electrophysiological techniques or radioactive ligand binding assays. The predictive power of these methods is assessed by comparing the results with the predictions that can be made on the basis of the recently published crystal structure of the acetylcholine binding protein that shows homology to the N-terminal, extracellular domain of the GABA(A) receptor. In addition, we review an approach to the question of how the benzodiazepine ligands are positioned in their binding pocket. We also discuss a newly postulated modulatory site for benzodiazepines at the α(1)/β(2) subunit interface, homologous to the classical benzodiazepine binding pocket.

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

Vascular endothelial growth factor: Crystal structure and functional mapping of the kinase domain receptor binding site

Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor β2 (TGF-β). We have determined its crystal structure at a resolution of 2.5 Å, and identified its kinase domain receptor (KDR) binding site using mutational analysis. Overall, the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is similar to that of PDGF and very different from that of TGF-β. Mutational analysis of VEGF reveals that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer. Each site contains two functional “hot spots” composed of binding determinants presented across the subunit interface. The two most important determinants are located within the largest hot spot on a short, three-stranded sheet that is conserved in PDGF and TGF-β. Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots.

Ordered water molecules as key allosteric mediators in a cooperative dimeric hemoglobin

One of the most remarkable structural aspects of Scapharca dimeric hemoglobin is the disruption of a very well-ordered water cluster at the subunit interface upon ligand binding. We have explored the role of these crystallographically observed water molecules by site-directed mutagenesis and osmotic stress techniques. The isosteric mutation of Thr-72 → Val in the interface increases oxygen affinity more than 40-fold with a surprising enhancement of cooperativity. The only significant structural effect of this mutation is to destabilize two ordered water molecules in the deoxy interface. Wild-type Scapharca hemoglobin is strongly sensitive to osmotic conditions. Upon addition of glycerol, striking changes in Raman spectrum of the deoxy form are observed that indicate a transition toward the liganded form. Increased osmotic pressure, which lowers the oxygen affinity in human hemoglobin, raises the oxygen affinity of Scapharca hemoglobin regardless of whether the solute is glycerol, glucose, or sucrose. Analysis of these results provides an estimate of six water molecules lost upon oxygen binding to the dimer, in good agreement with eight predicted from crystal structures. These experiments suggest that the observed cluster of interfacial water molecules plays a crucial role in communication between subunits.

Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.

Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.

Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia

Henneguya lesteri n. sp, (Myxosporea) is described from sand whiting, Sillago analis, from the southern Queensland coast of Australia. H. lesteri displays a preference for the pseudobranchs and is typically positioned along the afferent blood vessels, displacing the adjoining lamellae and disrupting their normal array, The plasmodia appeared as whitish-hyaline, elliptical cysts (mean dimensions 230 x 410 mum) attached to the oral mucosa lining of the hyoid arch on the inner surface of the operculum. Infections of the gills were also found, in which the plasmodia were spherical, averaged 240 x 240 mum in size and were located on the inner hemibranch margin. The parasites lodged in the gill filament crypts and generated a mild hyperplastic response of the branchial epithelium, In histological sections, the plasmodium wall and adjoining ectoplasm appeared as a finely granulated, weakly eosinophilic layer, Ultrastructurally, this section of the host-parasite interface contained an intricate complex of pinocytotic channels. H. lesteri is polysporic, disporoblastic and pansporoblast forming. Sporogenesis is asynchronous, with the earliest developmental stages aligned predominantly along the plasmodium periphery, and maturing sporoblasts and spores toward the center. Ultrastructural details of sporoblast and spore development are in agreement with previously described myxosporeans. The mature spore is drop-shaped, length (mean) 9.1 mum, width 4.7 mum, thickness 2.5 mum, and comprises 2 polar capsules positioned closely together, a binucleated sporoplasm and a caudal process of 12.6 mum. The polar capsules are elongated, 3.2 x 1.6 mum, with 4 turns of the polar filament. Mean length of the everted filament is 23.2 mum, Few studies have analyzed the 18S gene-of marine Myxosporea. In fact, H. lesteri is the first marine species of Henneguya to be characterized at the molecular level: we determined 1966 bp of the small-subunit (18S) rDNA, The results indicated that differences between this and the hitherto studied freshwater Henneguya species are greater than differences among the freshwater Henneguya species.

Light-activated rhodopsin induces structural binding motif in G protein α subunit

A large superfamily of transmembrane receptors control cellular responses to diverse extracellular signals by catalyzing activation of specific types of heterotrimeric GTP-binding proteins. How these receptors recognize and promote nucleotide exchange on G protein α subunits to initiate signal amplification is unknown. The three-dimensional structure of the transducin (Gt) α subunit C-terminal undecapeptide Gtα(340–350) IKENLKDCGLF was determined by transferred nuclear Overhauser effect spectroscopy while it was bound to photoexcited rhodopsin. Light activation of rhodopsin causes a dramatic shift from a disordered conformation of Gtα(340–350) to a binding motif with a helical turn followed by an open reverse turn centered at Gly-348, a helix-terminating C capping motif of an αL type. Docking of the NMR structure to the GDP-bound x-ray structure of Gt reveals that photoexcited rhodopsin promotes the formation of a continuous helix over residues 325–346 terminated by the C-terminal helical cap with a unique cluster of crucial hydrophobic side chains. A molecular mechanism by which activated receptors can control G proteins through reversible conformational changes at the receptor–G protein interface is demonstrated.

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment

The aa3 type cytochrome c oxidase consisting of the core subunits I and II only was isolated from the soil bacterium Paracoccus denitrificans and crystallized as complex with a monoclonal antibody Fv fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-β-d-maltoside and cyclohexyl-hexyl-β-d-maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P212121 and diffract x-rays to at least 2.5 Å (1 Å = 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 Å using molecular replacement and refined to a crystallographic R-factor of 20.5% (Rfree = 25.9%). The refined model includes subunits I and II and the 2 chains of the Fv fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg/Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. Only four of the water molecules are located in the cytoplasmic half of cytochrome c oxidase. Most of them are near the interface of subunits I and II. Several waters form a hydrogen-bonded cluster, including the heme propionates and the Mg/Mn binding site. The Fv fragment binds to the periplasmic polar domain of subunit II and is critically involved in the formation of the crystal lattice. The crystallization procedure is well reproducible and will allow for the analysis of the structures of mechanistically interesting mutant cytochrome c oxidases.

Subunit-destabilizing mutations in Drosophila copper/zinc superoxide dismutase: neuropathology and a model of dimer dysequilibrium.

Mutations in Cu/Zn superoxide dismutase (SOD), a hallmark of familial amyotrophic lateral sclerosis (FALS) in humans, are shown here to confer striking neuropathology in Drosophila. Heterozygotes with one wild-type and one deleted SOD allele retain the expected 50% of normal activity for this dimeric enzyme. However, heterozygotes with one wild-type and one missense SOD allele show lesser SOD activities, ranging from 37% for a heterozygote carrying a missense mutation predicted from structural models to destabilize the dimer interface, to an average of 13% for several heterozygotes carrying missense mutations predicted to destabilize the subunit fold. Genetic and biochemical evidence suggests a model of dimer dysequilibrium whereby SOD activity in missense heterozygotes is reduced through entrapment of wild-type subunits into unstable or enzymatically inactive heterodimers. This dramatic impairment of the activity of wild-type subunits in vivo has implications for our understanding of FALS and for possible therapeutic strategies.