Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope

We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC′ loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC′ loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.     

Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.     

Contrasting patterns of niche partitioning between two anaerobic terminal oxidizers of organic matter

Understanding the ecological principles underlying the structure and function of microbial communities remains an important goal for microbial ecology. We examined two biogeochemically important taxa, the sulfate-reducing bacterial genus, Desulfobulbus, and the methanogenic archaeal genus, Methanosaeta, to compare and contrast niche partitioning by these two taxa that are ecologically linked as anaerobic terminal oxidizers of organic material. An observational approach utilizing functional gene pyrosequencing was combined with a community-based reciprocal incubation experiment and characterization of a novel Desulfobulbus isolate. To analyze the pyrosequencing data, we constructed a data analysis pipeline, which we validated with several control data sets. For both taxa, particular genotypes were clearly associated with certain portions of an estuarine gradient, consistent with habitat or niche partitioning. Methanosaeta genotypes were generally divided between those found almost exclusively in the marine habitat (∼30% of operational taxonomic units (OTUs)), and those which were ubiquitously distributed across all or most of the estuary (∼70% of OTUs). In contrast to this relatively monotonic distribution, for Desulfobulbus, there were many more genotypes, and their distributions represented a wide range of inferred niche widths from specialist genotypes found only at a single site, to ubiquitous or generalist genotypes found in all 10 sites examined along the full estuarine gradient. Incubation experiments clearly showed that, for both taxa, communities from opposite ends of the estuary did not come to resemble one another, regardless of the chemical environment. Growth of a Desulfobulbus isolated into pure culture indicated that the potential niche of this organism is significantly larger than the realized niche. We concluded that niche partitioning can be an important force structuring microbial populations, with biotic and abiotic components having very different effects depending on the physiology and ecology of each taxon.     

Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair

Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.     

19F NMR investigation of molecular motion and packing in sonicated phospholipid vesicles

Dimyristoylphosphatidylcholine (DMPC) labeled with a C19F2 group in the 4-, 8-, or 12-position of the 2-acyl chain has been investigated in sonicated unilamellar vesicles (SUV) by fluorine-19 nuclear magnetic resonance (NMR) at 282.4 MHz from 26 to 42 degrees C. The 19F NMR spectra exhibit two overlapping resonances with different line widths. Spin-lattice relaxation time measurements have been performed in both the laboratory frame (T1) and the rotating frame (T1 rho) in order to investigate the packing and dynamics of phospholipids in lipid bilayers. Quantitative line-shape and relaxation analyses are possible by using the experimental chemical shift anisotropy (delta nu CSA) and the internuclear F-F vector order parameter (SFF) values obtained from the 19F powder spectra of multilamellar liposomes. The following conclusions can be made: The 19F chemical shift difference between the inside and outside leaflets of SUV can be used to monitor the lateral packing of the phospholipid in the two SUV monolayers. The hydrocarbon chains in the outer layer are found to be more tightly packed than those of the inner one, and the differences between them become smaller near the chain terminals. The effective correlation time [(1-4) x 10(-7) s] obtained from either the motional narrowing of the line widths or off-resonance T1 rho measurements is shorter than that estimated from the Stokes-Einstein diffusion model (10(-6) s), on the basis of a hydrodynamic radius of 110 A for SUV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of soluble pig heart glutamate-aspartate transaminase with various beta,gamma-unsaturated amino acids

beta-Ethylidene-DL-aspartate (beta EA) and beta-methylene-DL-glutamate (beta MG) were synthesized and tested as potential suicide inhibitors of soluble pig heart glutamate-aspartate transaminase (sGAT). beta MG was found to be a) a substrate with a very low turnover number relative to glutamate and b) a competitive inhibitor with respect to aspartate (albeit with a large binding constant). At high concentrations beta MG inactivated the enzyme but only very slowly. beta EA was also found to be a substrate with a very low turnover number; it did not inactivate the enzyme (1 hr, 25 degrees C) even at a high concentration. However, beta EA was found to bind to the enzyme with an affinity comparable to that of aspartate and glutamate. beta-Methylene-DL-aspartate (beta MA) has been shown to rapidly inactivate glutamate-aspartate transaminase. Therefore, it appears that glutamate-aspartate transaminase can bind analogues of aspartate with alkene groups in the beta position. The conjugated carbonyl groups of beta MA and beta EA will enhance Michael addition in comparison with that expected for vinylglycine. On the other hand, the presence of the methyl groups should reduce the electrophilicity of the double bond of beta EA compared to beta MA. This deactivation and/or steric hindrance to Michael attack may account for the inability of beta EA to inactivate sGAT. Therefore, it may be possible to design selective suicide inhibitors of glutamate-aspartate++ transaminase with the following structure: HO2CC(= CHX)CH(CO2H)NH2, where X is an electron-withdrawing group. Ideally, X would increase the reactivity of the double bond while affording a minimum of steric hindrance to susceptible enzyme-bound bases.