Gly Mutation Research Articles

Analysis of the Fluoroquinolone Antibiotic Resistance Mechanism of Salmonella enterica Isolates.

Quinolone-resistant Salmonella strains were isolated from patient samples, and several quinolone-sensitive strains were used to analyze mutations in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE and to screen for plasmid-mediated quinolone resistance. Among the 21 strains that showed resistance to nalidixic acid and ciprofloxacin (MIC 0.125-2.0 μg/ml), 17 strains had a mutation in QRDR codon 87 of gyrA, and 3 strains had a single mutation (Ser83 → Phe). Another cause of resistance, efflux pump regulation, was studied by examining the expression of acrB, ramA, marA, and soxS. Five strains, including Sal-KH1 and Sal-KH2, showed no increase in relative expression in an analysis using the qRT-PCR method (p < 0.05). In order to determine the genes involved in the resistance, the Sal-9 isolate that showed decreased susceptibility and did not contain a mutation in the gyrA QRDR was used to make the STM (MIC 8 μg/ml) and STH (MIC 16 μg/ml) ciprofloxacin-resistant mutants. The gyrA QRDR Asp87 → Gly mutation was identified in both the STM and STH mutants by mutation analysis. qRT-PCR analysis of the efflux transporter acrB of the AcrAB-TolC efflux system showed increased expression levels in both the STM (1.79-fold) and STH (2.0-fold) mutants. In addition, the expression of the transcriptional regulator marA was increased in both the STM (6.35-fold) and STH (21.73-fold) mutants. Moreover, the expression of soxS was increased in the STM (3.41-fold) and STH (10.05-fold) mutants (p < 0.05). Therefore, these results indicate that AcrAB-TolC efflux pump activity and the target site mutation in gyrA are involved in quinolone resistance.

Mapping the Effect of Gly Mutations in Collagen on α2β1 Integrin Binding

The replacement of one Gly in the essential repeating tripeptide sequence of the type I collagen triple helix results in the dominant hereditary bone disorder osteogenesis imperfecta. The mechanism leading to pathology likely involves misfolding and autophagy, although it has been hypothesized that some mutations interfere with known collagen interactions. Here, the effect of Gly replacements within and nearby the integrin binding GFPGER sequence was investigated using a recombinant bacterial collagen system. When a six-triplet human type I collagen sequence containing GFPGER was introduced into a bacterial collagen-like protein, this chimeric protein bound to integrin. Constructs with Gly to Ser substitutions within and nearby the inserted human sequence still formed a trypsin-resistant triple helix, suggesting a small local conformational perturbation. Gly to Ser mutations within the two Gly residues in the essential GFPGER sequence prevented integrin binding and cell attachment as predicted from molecular dynamics studies of the complex. Replacement of Gly residues C-terminal to GFPGER did not affect integrin binding. In contrast, Gly replacements N-terminal to the GFPGER sequence, up to four triplets away, decreased integrin binding and cell adhesion. This pattern suggests either an involvement of the triplets N-terminal to GFPGER in initial binding or a propagation of the perturbation of the triple helix C-terminal to a mutation site. The asymmetry in biological consequences relative to the mutation site may relate to the observed pattern of osteogenesis imperfecta mutations near the integrin binding site.

Structural Analysis of the Streptomyces avermitilis CYP107W1-Oligomycin A Complex and Role of the Tryptophan 178 Residue.

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of 2.6 Å. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant’s catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.

Abstract 12805: Postnatal Progressive Atrioventricular Block By Heterozygous Nkx2-5 Homeodomain Missense Mutation With Atrioventricular Nodal Atrophy

Introduction: Heterozygous human NKX2-5 homeodomain (DNA binding domain) missense mutations are highly penetrant for varied cardiac anomalies and progressive atrioventricular block (AVB). We replicated many elements of the cardiac anomalies in a murine heterozygous knock-in model (Nkx2-5+/R52G, hereafter Nkx2-5-KI) in a 129/Sv background. Hypothesis: We hypothesized that Nkx2-5-KI mice demonstrate progressive AVB during postnatal life similar to human patients. Methods: Nkx2-5-KI mouse model and control Nkx2-5+/+ mice were analyzed by histopathology, surface and telemetry ECG, and in vivo electrophysiology studies (EPS), and compared at postnatal day 1 (P1) to 17 months. We re-evaluated a pedigree of a human family having Arg52(189)Gly mutation in 5 generations. Results: Nkx2-5-KI mice demonstrated PR-prolongation (1st degree AVB) at 4 weeks, 7 months, and 17 months of age but not at P1 (control n=35, mutant n=38). Advanced AVB was occasionally observed. EPS showed that AV node function, and right ventricular effective refractory period were impaired in Nkx2-5-KI mice, while sinus node function was normal. AV node size was significantly smaller in Nkx2-5-KI mice relative to control mice (maximum area size 2.4 ± 0.2 vs. 1.6 ± 0.1 x 10-2 mm2, 36% reduction; calculated volume 1.50 ± 0.1 vs. 1.11 ± 0.03 x 10-4 mm3, 24% reduction, n=6 each) at 4 weeks of age but not P1, similar to age-dependence of PR-prolongation. AV node in mutant mice was composed of smaller cardiomyocytes without fibrosis, suggesting in part, that conduction abnormalities are the result of an AV node that becomes morphologically atrophic during postnatal life. All 6 genotype-positive humans demonstrated AVB (1st, 2nd, and 3rd degree). QRS prolongation was uniquely demonstrated in Nkx2-5-KI mice from the neonatal stage onward but not in human patients, suggesting substantial discordance in ventricular conduction system between mouse and human. Conclusion: Highly penetrant and progressive AVB similar to that seen in humans with heterozygous NKX2-5 homeodomain missense mutations was replicated in mice by knocking-in a comparable missense mutation. The progressive AVB phenotype is likely due to an AV node composed of smaller cardiomyocytes that becomes atrophic during postnatal life.

A Natural Interruption Displays Higher Global Stability and Local Conformational Flexibility than a Similar Gly Mutation Sequence in Collagen Mimic Peptides.

Natural interruptions in the repeating (Gly-X-Y)n amino acid sequence pattern are found normally in triple helix domains of all nonfibrillar collagens, while any Gly substitution in fibrillar collagens leads to pathological conditions. As revealed by our sequence analysis, two peptides, one modeling a natural G5G interruption (POALO) and the other one mimicking a pathological Gly-to-Ala substitution (LOAPO), are designed. Circular dichroism (CD), NMR, and computational simulation studies have discovered significant differences in stability, conformation, and folding between the two peptides. Compared with the Gly substitution sequence, the natural interruption maintains higher stability, higher triple helix content, and a higher folding rate while introducing more alterations in local triple helical conformation in terms of dihedral angles and hydrogen bonding. The conserved hydrophobic residues at the specific sites of interruptions may provide functional constraints for higher-order assembly as well as biomolecular interactions. These results suggest a molecular basis of different biological roles of natural interruptions and Gly substitutions and may guide the design of collagen mimic peptides containing functional natural interruptions.

Field-evolved resistance to four modes of action of herbicides in a single kochia (Kochia scoparia L. Schrad.) population.

Evolution of multiple herbicide resistance in weeds is a serious threat to weed management in crop production. Kochia is an economically important broadleaf weed in the U.S. Great Plains. This study aimed to confirm resistance to four sites of action of herbicides in a single kochia (Kochia scoparia L. Schrad.) population from a crop field near Garden City (GC), Kansas, and further determine the underlying mechanisms of resistance. One-fourth of the GC plants survived the labeled rate or higher of atrazine [photosystem II (PSII) inhibitor], and the surviving plants had the Ser-264 to Gly mutation in the psbA gene, the target site of atrazine. Results showed that 90% of GC plants survived the labeled rate of dicamba, a synthetic auxin. At least 87% of the plants survived up to 72 g a.i. ha(-1) of chlorsulfuron [acetolactate synthase (ALS) inhibitor], and analysis of the ALS gene revealed the presence of Pro-197 to Thr and/or Trp-574 to Lue mutation(s). Most GC plants also survived the labeled rate of glyphosate [5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor), and the resistant plants had 5-9 EPSPS gene copies (relative to the ALS gene). We confirm the first case of evolution of resistance to four herbicide sites of action (PSII, ALS and EPSPS inhibitors and synthetic auxins) in a single kochia population, and target-site-based mechanisms confer resistance to atrazine, glyphosate and chlorsulfuron.

Local amino acid sequence patterns dominate the heterogeneous phenotype for the collagen connective tissue disease Osteogenesis Imperfecta resulting from Gly mutations.

Osteogenesis Imperfecta (OI), a hereditary connective tissue disease in collagen that arises from a single Gly → X mutation in the collagen chain, varies widely in phenotype from perinatal lethal to mild. It is unclear why there is such a large variation in the severity of the disease considering the repeating (Gly-X-Y)n sequence and the uniform rod-like structure of collagen. We systematically evaluate the effect of local (Gly-X-Y)n sequence around the mutation site on OI phenotype using integrated bio-statistical approaches, including odds ratio analysis and decision tree modeling. We show that different Gly → X mutations have different local sequence patterns that are correlated with lethal and nonlethal phenotypes providing a mechanism for understanding the sensitivity of local context in defining lethal and non-lethal OI. A number of important trends about which factors are related to OI phenotypes are revealed by the bio-statistical analyses; most striking is the complementary relationship between the placement of Pro residues and small residues and their correlation to OI phenotype. When Pro is present or small flexible residues are absent nearby a mutation site, the OI case tends to be lethal; when Pro is present or small flexible residues are absent further away from the mutation site, the OI case tends to be nonlethal. The analysis also reveals the dominant role of local sequence around mutation sites in the Major Ligand Binding Regions that are primarily responsible for collagen binding to its receptors and shows that non-lethal mutations are highly predicted by local sequence considerations alone whereas lethal mutations are not as easily predicted and may be a result of more complex interactions. Understanding the sequence determinants of OI mutations will enhance genetic counseling and help establish which steps in the collagen hierarchy to target for drug therapy.

Reduced Culture Temperature Differentially Affects Expression and Biophysical Properties of Monoclonal Antibody Variants.

Reduced culture temperature is an increasingly popular practice to improve recombinant protein yields in CHO cells. Recent studies have attributed the enhancement of protein titers at sub-physiological temperatures to increased mRNA levels as well as extended stationary phase. We observed that reducing the culture temperature arrested cell growth, prolonged viability, and increased cell size. However, the reduced culture temperature had a differential effect on protein and mRNA expression of closely related antibody mutants from stable cell lines. The highly expressing mutant (Ala) exhibited similar or decreased specific productivity and decreased volumetric productivity over the culture lifetime at 32 °C compared to 37 °C. In contrast, the specific and volumetric productivity of the poorly expressing mutant (Gly) was enhanced at the lower culture temperature. The difference in specific productivity was reflected in the amounts of heavy- and light-chain mRNA. Analysis of the secondary and tertiary configurations of the purified antibodies by circular dichroism revealed fundamental structural differences imposed by the Ala to Gly mutation as well as reduced culture temperature. We propose that the effect of reduced culture temperature on expression is protein-dependent; protein folding fidelity and assembly is improved at lower temperatures, enhancing the expression of proteins that have a propensity to misfold.

Perturbations of the Straight Transmembrane α-Helical Structure of the Amyloid Precursor Protein Affect Its Processing by γ-Secretase

The amyloid precursor protein (APP) is a widely expressed type I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by γ-secretase of its C-terminal fragment produces amyloid-β (Aβ) peptides of different lengths, the deposition of which is an early indicator of Alzheimer disease. At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by NMR in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight α-helical conformation once embedded in a membrane bilayer. However, APP-TM is highly flexible, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance spectroscopy. These structural and dynamic features are critical in the proteolytic processing of APP by the γ-secretase enzyme, as suggested by a series of Gly(700) mutants. Affecting the hydration and flexibility of APP-TM, these mutants invariantly show an increase in the production of Aβ38 compared with Aβ40 peptides, which is reminiscent of the effect of γ-secretase modulators inhibitors.

Characterization of a Galactosynthase Derived from Bacillus circulans β‐Galactosidase: Facile Synthesis of D‐Lacto‐ and D‐Galacto‐N‐bioside

Glycosynthases-retaining glycosidases mutated at their catalytic nucleophile-catalyze the formation of glycosidic bonds from glycosyl fluorides as donor sugars and various glycosides as acceptor sugars. Here the first glycosynthase derived from a family 35 β-galactosidase is described. The Glu→Gly mutant of BgaC from Bacillus circulans (BgaC-E233G) catalyzed regioselective galactosylation at the 3-position of the sugar acceptors with α-galactosyl fluoride as the donor. Transfer to 4-nitophenyl α-D-N-acetyl-glucosaminide and α-D-N-acetylgalactosaminide yielded 4-nitophenyl α-lacto-N-biose and α-galacto-N-biose, respectively, in high yields (up to 98%). Kinetic analysis revealed that the high affinity of the acceptors contributed mostly to the BgaC-E233G-catalyzed transglycosylation. BgaC-E233G showed no activity with β-(1,3)-linked disaccharides as acceptors, thus suggesting that this enzyme can be used in "one-pot synthesis" of LNB- or GNB-containing glycans.

The Periplasmic Cavity of LacY Mutant Cys154→Gly: How Open Is Open?

The lactose permease from Escherichia coli (LacY) is a galactoside/H(+) symporter that catalyzes the coupled stoichiometric transport of a sugar and an H(+) across the cytoplasmic membrane. X-ray crystal structures of WT LacY and the conformationally restricted mutant Cys154→Gly exhibit an inward-facing conformation with a tightly sealed periplasmic side and a deep central cleft or cavity open to the cytoplasm. Although the crystal structures may give the impression that LacY is a rigid molecule, multiple converging lines of evidence demonstrate that galactoside binding to WT LacY induces reciprocal opening and closing of periplasmic and cytoplasmic cavities, respectively. By this means, the sugar- and H(+)-binding sites in the middle of the molecule are exposed alternatively to either side of the membrane. In contrast to the crystal structure, biochemical/biophysical studies with mutant Cys154→Gly show that the periplasmic side is paralyzed in an open-outward conformation. In this study, a rigid, funnel-shaped, maleimide-containing molecule was used to probe the periplasmic cavity of a pseudo-WT and the Cys154→Gly mutant by site-directed alkylation. The findings provide strong support for previous observations and indicate further that the external opening of the periplasmic cleft in the mutant is patent to the extent of at least 8.5 Å in the absence of sugar or about half that of the WT cavity with bound galactoside.

Ala-7, His-10 and Arg-12 are crucial amino acids for activity of a synthetically engineered μ-conotoxin

Cone snail toxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels such as voltage-gated sodium channels (NaVs). NaVs participate in a wide range of electrophysiological processes. Consequently, their malfunction has been associated with numerous diseases. The development of subtype-selective modulators of NaVs remains highly important in the treatment of such disorders. In order to expand our knowledge in the search for novel therapeutics to treat NaV-related diseases, we explored the field of peptide engineering. In the current study, the impact of well considered point mutations into a bioactive peptide that was found to be a very potent and selective inhibitor of NaVs (i.e. Midi R2) was examined. We designed two peptides, named Midi R2[A7G] and Midi R2[H10A, R12A] which have mutations at position 7, and both 10 and 12, respectively. Electrophysiological recordings indicated that an Ala to Gly mutation at position 7 increased IC50-values from the nanomolar range to the micromolar range. For Midi R2[H10A, R12A] at a concentration of 10μM, activity is even reduced to 0–10% for all of the tested NaV-channels. Circular dichroism measurements proved that overall structural conformations did not change. These findings suggest that the minimal space between the second and the third intercysteine loop of Midi R2 is the sequence RRWARDHSR and that His at position 10 and Arg at position 12 are crucial amino acids for the potency and specificity of Midi R2. In this way, new insights into the structure–activity relationships of μ-conotoxins were found.

Protein engineering of oxidosqualene-lanosterol cyclase into triterpene monocyclase

A computational modeling/protein engineering approach was applied to probe H234, C457, T509, Y510, and W587 within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7), which spatially affects the C-10 cation of lanosterol formation. Substitution of Trp587 to aromatic residues supported the "aromatic hypothesis" that the π-electron-rich pocket is important for the stabilization of electron-deficient cationic intermediates. The Cys457 to Gly and Thr509 to Gly mutations disrupted the pre-existing H-bond to the protonating Asp456 and the intrinsic His234 : Tyr510 H-bond network, respectively, and generated achilleol A as the major product. An H234W/Y510W double mutation altered the ERG7 function to achilleol A synthase activity and generated achilleol A as the sole product. These results support the concept that a few-ring triterpene synthase can be derived from polycyclic cyclases by reverse evolution, and exemplify the power of computational modeling coupled with protein engineering both to study the enzyme's structure-function-mechanism relationships and to evolve new enzymatic activity.

Triazine-Resistant Annual Bluegrass (Poa annua) Populations with Ser264Mutation Are Resistant to Amicarbazone

Amicarbazone is a photosystem II (PSII)-inhibiting herbicide in the triazolinone family, which is similar in mode of action to the triazines. Annual bluegrass is a cool-season weed and has shown resistance to some PSII-inhibiting herbicides. The objective was to evaluate triazine-resistant and -susceptible annual bluegrass populations for potential cross-resistance to amicarbazone. Two triazine-resistant (MS-01, MS-02) and triazine-susceptible (AL-01, COM-01) annual bluegrass populations were treated with amicarbazone, atrazine, and simazine at 0.26, 1.7, and 1.7 kg ai ha−1, respectively. All herbicide treatments controlled the susceptible populations greater than 94% 2 wk after treatment (WAT). No visual injury of MS-01 and MS-02 was observed at any time following herbicide treatment. Quantum yield (ΦPSII) of annual bluegrass was measured 0 to 72 h after application (HAA) to determine the photochemical effects of amicarbazone compared to other PSII inhibitors. ΦPSIIof triazine-susceptible populations was reduced at all measurement times by all three herbicides. However, amicarbazone decreased ΦPSIIof susceptible populations faster and greater than atrazine and simazine at most measurement times. Amicarbazone did not reduce ΦPSIIof the MS-01 population. Amicarbazone significantly reduced ΦPSIIof the MS-02 population during several measurement timings; however, these reductions were short-lived compared to the susceptible populations and no trend in ΦPSIIreduction was observed. Sequencing of thepsbAgene revealed a Ser to Gly substitution at amino acid position 264 known to confer resistance to triazine herbicides. These data indicate amicarbazone efficiently inhibited PSII of susceptible annual bluegrass populations; however, triazine-resistant annual bluegrass populations with Ser264to Gly mutations are cross-resistant to amicarbazone.

Dissecting the function of a protruding loop in AcrB trimerization

The resistance-nodulation-cell division family multidrug transporter Acriflavine resistance protein B (AcrB) from Eschericha coli is an obligate homotrimer. Approximately, 45% of the inter-subunit interface is contributed by a protruding loop (also referred to as the thumb) and its corresponding binding tunnel in the neighboring subunit. In an earlier study, we have demonstrated that a single Pro to Gly mutation in the loop drastically destabilized AcrB trimer and reduced its substrate efflux activity. To further dissect the role of the loop during AcrB trimerization, we performed Ala scanning of the loop and examined the effect of each mutation on protein activity. We found that not all conserved residues are important for AcrB function and likewise not all critical residues are conserved. In addition, we replaced the loop of AcrB with the loop of MexB, which is a highly conserved homolog of AcrB. The resultant chimeric protein remained partly active. Structural characterization of the chimeric protein indicated that it was well folded and existed as a mixture of monomer and trimer. Our results indicate that the loop to tunnel interaction, while critical to trimerization and efflux function, is in general rather flexible and tolerant to mutations. In addition, all mutations in the loop that resulted in reduced function clustered closely, suggesting that this may be the site of inter-subunit recognition during trimerization and/or a locking zone to stabilize the inter-subunit interaction during trimerization.

Site E14 in Hemoglobins and Myoglobins: A Key Residue That Affects Hemin Loss and Lipid Oxidation Capacity

Fish hemoglobins (Hbs) frequently contain glycine at site E14 while mammalian Hbs contain larger residues (e.g., alanine and serine). These differences were examined by creating structural variants at E14 using recombinant bovine myoglobin (Mb) as a model heme protein that contains alanine at E14. The Ala(E14)Gly mutation increased k(ox) and hemin loss 3-fold and 45-fold, respectively. Glycine at E14 creates a channel for solvent to enter the heme crevice, which enhances autoxidation and hemin loss rates. Hydration of the proximal heme pocket facilitates hemin loss because protonation of the proximal histidine weakens the linkage of the imidazole group to the iron atom of the hemin moiety. Ala(E14)Gly promoted lipid oxidation in washed fish muscle more rapidly during iced storage compared to wild type Mb at pH 5.7. This suggested that the rapid hemin loss from Ala(E14)Gly accelerated lipid oxidation. Ala(E14)Ser and Ala(E14)Val had little effect on k(ox) but somewhat accelerated net hemin loss. These studies suggest that enhanced access of solvent to the heme crevice of many fish Hbs at site E14 facilitates rapid hemin loss and moderately accelerates autoxidation. This likely is part of the reason fish Hbs promote lipid oxidation much more effectively compared to mammalian Hbs.

Discovery of a Novel Allosteric Modulator of 5-HT3 Receptors: INHIBITION AND POTENTIATION OF CYS-LOOP RECEPTOR SIGNALING THROUGH A CONSERVED TRANSMEMBRANE INTERSUBUNIT SITE

The ligand-gated ion channels in the Cys-loop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA, and glycine. Cys-loop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel class of negative allosteric modulators of the 5-HT(3) receptors (5-HT(3)Rs). PU02 (6-[(1-naphthylmethyl)thio]-9H-purine) is a potent and selective antagonist displaying IC(50) values of ~1 μM at 5-HT(3)Rs and substantially lower activities at other Cys-loop receptors. In an elaborate mutagenesis study of the 5-HT(3)A receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)-subunit and TM1 and TM2 in the (-)-subunit. The Ser(248), Leu(288), Ile(290), Thr(294), and Gly(306) residues are identified as important molecular determinants of PU02 activity with minor contributions from Ser(292) and Val(310), and we propose that the naphthalene group of PU02 docks into the hydrophobic cavity formed by these. Interestingly, specific mutations of Ser(248), Thr(294), and Gly(306) convert PU02 into a complex modulator, potentiating and inhibiting 5-HT-evoked signaling through these mutants at low and high concentrations, respectively. The PU02 binding site in the 5-HT(3)R corresponds to allosteric sites in anionic Cys-loop receptors, which emphasizes the uniform nature of the molecular events underlying signaling through the receptors. Moreover, the dramatic changes in the functional properties of PU02 induced by subtle changes in its binding site bear witness to the delicate structural discrimination between allosteric inhibition and potentiation of Cys-loop receptors.

Analysis of local spread of metamitron‐resistant Chenopodium album patches in Belgium

Aper J, Mechant E, De Riek J, Van Laere K, Bulcke R &amp; Reheul D (2012). Analysis of local spread of metamitron‐resistant Chenopodium album patches in Belgium. Weed Research52, 421–429.SummaryTracing spread of weeds with molecular markers can give valuable information on the importance of migration mechanisms. This study investigated the local spread of metamitron‐resistant Chenopodium album patches in the west of the province West Flanders (Belgium) using amplified fragment length polymorphism (AFLP) markers. During the summer of 2009, leaf samples of C. album plants were harvested in 27 patches, distributed over 10 sugar beet fields and one maize field. The fields were grouped in four local clusters, each corresponding to a farmer who cultivated these fields. A cleaved amplified polymorphic sequence procedure identified the Ser264 to Gly mutation in the D1 protein, endowing resistance to metamitron, a key herbicide applied in sugar beet. The majority of the sampled plants within a patch (97% on average) carried this mutation. Genetic variation among the four farmers’ locations (12%) and among the C. album patches within the farmers’ locations (14%) was significant according to amova (P &lt; 0.001). In addition, Mantel tests confirmed a positive correlation between genetic distance (linearised фPT between pairs of patches) and the logarithm of geographic distance for the complete data set (Mantel coefficient significant at P = 0.001), suggesting isolation by distance. Nevertheless, genetic similarity between patches from different fields indicated that seed transport by agricultural machinery and manure is likely to have an important impact on the spread of metamitron‐resistant biotypes. Farmers should become aware of the resistance problem as soon as possible, in order to prevent further spread in their fields.

Role of Conserved Glycine in Zinc-dependent Medium Chain Dehydrogenase/Reductase Superfamily

The medium-chain dehydrogenase/reductase (MDR) superfamily consists of a large group of enzymes with a broad range of activities. Members of this superfamily are currently the subject of intensive investigation, but many aspects, including the zinc dependence of MDR superfamily proteins, have not yet have been adequately investigated. Using a density functional theory-based screening strategy, we have identified a strictly conserved glycine residue (Gly) in the zinc-dependent MDR superfamily. To elucidate the role of this conserved Gly in MDR, we carried out a comprehensive structural, functional, and computational analysis of four MDR enzymes through a series of studies including site-directed mutagenesis, isothermal titration calorimetry, electron paramagnetic resonance (EPR), quantum mechanics, and molecular mechanics analysis. Gly substitution by other amino acids posed a significant threat to the metal binding affinity and activity of MDR superfamily enzymes. Mutagenesis at the conserved Gly resulted in alterations in the coordination of the catalytic zinc ion, with concomitant changes in metal-ligand bond length, bond angle, and the affinity (K(d)) toward the zinc ion. The Gly mutants also showed different spectroscopic properties in EPR compared with those of the wild type, indicating that the binding geometries of the zinc to the zinc binding ligands were changed by the mutation. The present results demonstrate that the conserved Gly in the GHE motif plays a role in maintaining the metal binding affinity and the electronic state of the catalytic zinc ion during catalysis of the MDR superfamily enzymes.

Evidence for an intermediate conformational state of LacY

LacY mutant Cys154 → Gly exhibits a periplasmic-closed crystal structure identical to the WT, but is periplasmic-open in the membrane. The mutant hardly catalyzes transport, but binds galactosides from either side of the membrane with the same affinity and is resistant to site-directed proteolysis relative to the pseudo-WT. Site-directed alkylation was also applied to 11 single-Cys mutants in Cys154 → Gly LacY in right-side-out membrane vesicles or after solubilization and purification in dodecyl-β-D-maltopyranoside (DDM). Unlike the pseudo-WT, Cys replacements on the periplasmic side of the Cys154 → Gly mutant label rapidly in the membrane without sugar, but labeling decreases markedly after the mutant proteins are purified. Thus, Cys154 → Gly LacY likely favors a higher-energy intermediate periplasmic-open conformation in situ, but collapses to a lower-energy periplasmic-closed conformation in DDM after purification. Notably, branched-chain or neopentyl glycol maltoside detergents stabilize Cys154 → Gly LacY in the membrane-embedded form.