Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase

Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography. These are compared with the previously determined structure of magnesium and sulphate-bound XPRT. The structure of free XGPRT at 2.25 Angstrom resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands. The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 Angstrom resolution. In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5'-phosphate group of cPRib-PP. The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+. The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135. Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 .when the nucleotide product is formed. The 2.6 Angstrom resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes. However, the ribose ring of GMP is rotated by similar to 24 degrees compared with the equivalent ring in cPRib-PP. This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex. (C) 1998 Academic Press.

Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways

Molecular mechanisms of zinc potentiation were investigated in recombinant human alpha 1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [H-3]strychnine binding assays. In the wild-type (WT) GlyR, 1 mu M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1-M2 and M2-M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2-M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

A pore-size-dependent equation of state for multilayer adsorption in cylindrical mesopores

The chemical potential of adsorbed film inside cylindrical mesopores is dependent on the attractive interactions between the adsorbed molecules and adsorbent, the curvature of gas/adsorbed phase interface, and surface tension. A state equation of the adsorbed film is proposed to take into account the above factors. Nitrogen adsorption on model adsorbents, MCM-41, which exhibit uniform cylindrical channels, are used to verify the theoretical analysis. The proposed theory is capable of describing the important features of adsorption processes in cylindrical mesopores. According to this theory, at a given relative pressure, the smaller the pore radius is, the thicker the adsorbed film will be. The thickening of adsorbed films in the pores as the vapor pressure increases inevitably causes an increase in the interface curvature, which consequently leads to capillary condensation. Besides, this study confirmed that the interface tension depends substantially on the interface curvature in small mesopores. A quantitative relationship between the condensation pressure and the pore radius can be derived from the state equation and used to predict the pore radius from a condensation pressure, or vice versa.

H-1-NMR structural studies of a cystine-linked peptide containing residues 71-93 of transthyretin and effects of a Ser84 substitution implicated in familial amyloidotic polyneuropathy

The Ile-->Ser84 substitution in the thyroid hormone transport protein transthyretin is one of over 50 variations found to be associated with familial amyloid polyneuropathy, a hereditary type of lethal amyloidosis. Using a peptide analogue of the loop containing residue 84 in transthyretin, we have examined the putative local structural effects of this substitution using H-1-NMR spectroscopy. The peptide, containing residues 71-93 of transthyretin with its termini linked via a disulfide bond, was found to possess the same helix-turn motif as in the corresponding region of the crystallographically derived structure of transthyretin in 20% trifluoroethanol (TFE) solution. It therefore, represents a useful model with which to examine the effects of amyloidogenic substitutions. In a peptide analogue containing the Ile84-->Ser substitution it was found that the substitution does not greatly disrupt the overall three-dimensional structure, but leads to minor local differences at the turn in which residue 84 is involved. Coupling constant and NOE measurements indicate that the helix-turn motif is still present, but differences in chemical shifts and amide-exchange rates reflect a small distortion. This is in keeping with observations that several other mutant forms of transthyretin display similar subunit interactions and those that have been structurally analysed possess a near native structure. We propose that the Ser84 mutation induces only subtle perturbations to the transthyretin structure which predisposes the protein to amyloid formation.

The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: Fivefold tandem repetition of a coding region

We sequenced across all of the gene boundaries in the mitochondrial genome of the cattle tick, Boophilus microplus, to determine the arrangement of its genes. The mtDNA of B. microplus has a coding region, composed of tRNA(Glu) and 60 bp of the 3' end of ND1, that is repeated five times. Boophilus microplus is the first coelomate animal known to have more than two copies of a coding sequence. The mitochondrial genome of B, microplus has other unusual features, including (1) reduced T arms in tRNAs, (2) an AT bias in codon use, (3) two control regions that have evolved in concert, (4) three gene rearrangements, and (5) a stem-loop between tRNA(Gln) and tRNA(Phe). The short T arms and small control regions (CRs) of B. microplus and other ticks suggest strong selection for small genomes. Imprecise termination of replication beyond its origin, which can account for the evolution of tandem repeats of coding regions in other mitochondrial genomes, cannot explain the evolution of the fivefold repeated sequence in the mitochondrial genome of B. microplus. Instead, slipped-strand mispairing or recombination are the most plausible explanations for the evolution of these tandem repeats.

Solution structure of alpha-conotoxin ImI by H-1 nuclear magnetic resonance

alpha-Conotoxin ImI derives from the venom of Conus imperialis and is the first and only small-peptide ligand that selectively binds to the neuronal alpha(7) homopentameric subtype of the nicotinic acetylcholine receptor (nAChR). This receptor subtype is a possible drug target for several neurological disorders. The cysteines are connected in the pairs Cys2-Cys8 and Cys3-Cys12, To date it is the only alpha-conotoxin with a 4/3 residue spacing between the cysteines, The structure of ImI has been determined by H-1 NMR spectroscopy in aqueous solution, The NMR structure is of high quality, with a backbone pairwise rmsd of 0.34 Angstrom for a family of 19 structures, and comprises primarily a series of nested beta turns. Addition of organic solvent does not perturb the solution structure. The first eight residues of ImI are identical to the larger, but related, conotoxin EpI and adopt a similar structure, despite a truncated second loop. Residues important for binding of ImI to the alpha 7 nAChR are all clustered on one face of the molecule. Once further binding data for EPI and ImI are available, the ImI structure will allow for design of novel alpha(7) nAChR-specific agonists and antagonists with a wide range of potential pharmaceutical applications.

Control of fed-batch fermentations

Fed-batch fermentation is used to prevent or reduce substrate-associated growth inhibition by controlling nutrient supply. Here we review the advances in control of fed-batch fermentations. Simple exponential feeding and inferential methods are examined, as are newer methods based on fuzzy control and neural networks. Considerable interest has developed in these more advanced methods that hold promise for optimizing fed-batch techniques for complex fermentation systems. (C) 1999 Elsevier Science Inc. All rights reserved.

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene from Nicotiana alata

Background: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (Pls) that are derived from a 43 kDa precursor protein, NaProPl. NaProPl contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native Pls. An unusual feature of NaProPl is that the structural domains lie across adjacent repeats and that the sixth Pl domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPl repeat (aPl1) forms a basis for unravelling the mechanism by which this protein may have evolved, Results: The three-dimensional structure of aPl1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native Pls. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPl, exists as a disordered loop in aPl1. The presence of this loop in aPl1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. Conclusions: A single repeat from NaProPl is capable of folding into a compact globular domain that displays native-like Pl activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPl evolved.

Identification of a new ligand binding domain in the al subunit of the inhibitory glycine receptor

Four discontinuous extracellular sequence domains have been proposed to form the ligand binding sites of the ligand-gated ion channel receptor superfamily. In this study, we investigated the role of 12 contiguous residues of the inhibitory glycine receptor that define the proposed loop A ligand binding domain; Using the techniques of site-directed mutagenesis and patch-clamp electrophysiology, four of the 12 residues were shown to have impaired ligand binding. Three mutants, I93A, A101H, and N102A, resulted in significant (17-44-fold) increases in the agonist EC50 values as compared with the wild-type glycine receptor, whereas Hill coefficients, I-max values, and antagonist affinity remained largely unaffected. Consideration of receptor efficacy values indicates that these residues are involved in ligand binding rather than channel activation. A fourth mutant, W94A, failed to give rise to any glycine-activated currents, although cell-surface expression was observed, suggesting that this residue may also be involved in agonist binding. These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile(93) and Asn(102), as contributing to the four-loop model of ligand binding.

Identification of residues involved in binding of IL5 to beta(com) using beta(IL3) and beta(com) chimeras

In mice there are two forms of the beta chain used in the IL3 receptor system, beta(com) and beta(IL3). beta(com) is used by the IL3, ILS and GM-CSF receptors whereas Pns is only used in the IL3 receptor. In this work an assay was developed to identify residues of beta(IL3) that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the beta(IL3) B'-C' loop were replaced with beta(com) sequence a form of beta(IL3) was produced that was able to respond to IL5. This region is also responsible for IL3 binding to beta(IL3) in the absence of alpha chain. It is therefore an important structural motif of beta(com) and beta(IL3) responsible for both ligand interaction and specificity. (C) 1999 Federation of European Biochemical Societies.

Sulfite : Cytochrome c oxidoreductase from Thiobacillus novellus - Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alpha beta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytochrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K-m values for sulfite and cytochrome c(550) were determined to be 27 and 4 mu M, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

Reverse transcription of a naturally occurring nonretroviral RNA produces a precise deletion in the majority of its cDNA products

A precise, reproducible deletion made during in vitro reverse transcription of RNA2 from the icosahedral positive-stranded Helicoverpa armigera stunt virus (Tetraviridae) is described. The deletion, located between two hexamer repeats, is a 50-base sequence that includes one copy of the hexamer repeat. Only the Moloney murine leukemia virus reverse transcriptase and its derivative Superscript I, carrying a deletion of the carboxy-terminal RNase H region, showed this response, indicating a template-switching mechanism different from one proposed that involves a RNase H-dependent strand transfer, Superscript II, however, which carries point mutations to reduce RNase H activity, does not cause a deletion. A possible mechanism involves the enzyme pausing at the 3' side of a stem-loop structure and the 3' end of the nascent DNA strand separating from the template and reannealing to the upstream hexamer repeat.

Functional implications of the human T-lymphotropic virus type 1 transmembrane glycoprotein helical hairpin structure

Retrovirus entry into cells follows receptor binding by the surface exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.

Identification of regions within the third FnIII-like domain of the IL-5R alpha involved in IL-5 interaction

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5R alpha). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5R alpha (hIL-5R alpha) and mouse IL-5R alpha (mIL-5R alpha) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5R alpha but not the mIL-5R alpha. By using chimeras of the mIL-5R alpha and hIL-5R alpha we demonstrate that residues within the first and third FnIII-like domains of mIL-5R alpha are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5R alpha to mIL-5R alpha within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity, These results show that regions of the third FnIII domain of IL-5R alpha are involved in binding, in addition to the regions in domains one and two of the IL-5R alpha that were identified in an earlier study. (C) 2000 Academic Press.