Formation of a chloride-conducting state in the Maltose ATP-Binding Cassette (ABC) transporter [Protein Structure and Folding]

April 7th, 2016 by Carlson, M. L., Bao, H., Duong, F.

ABC transporters use an alternating access mechanism to move substrates across cellular membranes. This mode of transport ensures the selective passage of molecules while preserving membrane impermeability. The crystal structures of MalFGK2, inward- and outward-facing, show that the transporter is sealed against ions and small molecules. It has yet to be determined whether membrane impermeability is maintained when MalFGK2 cycles between these two conformations. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that not only is chloride conductance occurring, but also to a degree large enough to compromise cell viability. Introduction of mutations in the periplasmic gate lead to the formation of a channel that is quasi-permanently open. MalFGK2 must therefore stay away from these ion-conducting conformations in order to preserve the membrane barrier; otherwise, a few mutations that increase access to the ion-conducting states are enough to convert an ABC transporter into a channel.

DNA Polymerase III, but not Polymerase IV, Must be Bound to {tau}-Containing DnaX Complex to Enable Exchange into Replication Forks [Enzymology]

April 7th, 2016 by Yuan, Q., Dohrmann, P. R., Sutton, M. D., McHenry, C. S.

Examples of dynamic polymerase exchange have been previously characterized in the model systems provided by coliphages T4 and T7. Using a dominant negative D403E Pol III α that can form initiation complexes and sequester primer termini, but not elongate, we investigated the possibility of exchange at the E. coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a containing DnaX complex, did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of τ or the alternative shorter dnaX translation product γ, showed that τ1, τ2, or τ3 -DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size and gaps between fragments, possessed the ability to challenge pre-established replication forks and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one τ. Previously, it was thought that at least two τs in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed.
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Role of glyoxylate shunt in oxidative stress response [Microbiology]

April 1st, 2016 by Ahn, S., Jung, J., Jang, I.-A., Madsen, E. L., Park, W.

The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the TCA cycle. The GS bypasses the carbon dioxide-producing steps of the TCA cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be upregulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli. In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including upregulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer: a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress.

Structural Divergence of the Group I Intron-Binding Surface in Fungal Mitochondrial Tyrosyl-tRNA Synthetases that Function in RNA Splicing [RNA]

April 1st, 2016 by Lamech, L. T., Saoji, M., Paukstelis, P. J., Lambowitz, A. M.

The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mt tRNATyr and promote the splicing of autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon-binding domains and that the catalytic domain uses a newly evolved group I intron-binding surface, which includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA-binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined X-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the A. fumigatus mtTyrRS showed that the overall topology of the group I intron-binding surface is conserved, but with variations in key intron-binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions that also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron-specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi.
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Entry of Bluetongue virus capsid requires the late endosomal specific lipid lysobisphosphatidic acid [Lipids]

April 1st, 2016 by Patel, A., Mohl, B.-P., Roy, P.

The entry of viruses into host cells is one of the key processes for the infection.. The mechanisms of cellular entry for enveloped virus have been well studied. The fusion proteins as well as the facilitating cellular lipid factors involved in the viral fusion entry process have been well characterized. The process of non-enveloped virus cell entry, in comparison, remains poorly defined, particularly for large complex capsid viruses of the family Reoviridae, which comprises a range of mammalian pathogens. These viruses enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes to enter cells. Instead, these viruses are believed to penetrate membranes of the host cell during endocytosis. However, the molecular mechanism of this process is largely undefined. Here we show utilizing an in vitro liposome penetration assay and cell biology that Bluetongue virus (BTV), an archetypal member of the Reoviridae, utilizes the late endosomal specific lipid lysobisphosphatidic acid (LBPA) for productive membrane penetration and viral entry. Further we provide preliminary evidence that LBPA facilitates pore expansion during membrane penetration suggesting a mechanism for lipid factor requirement of BTV. This data indicates that despite the lack of a membrane envelope, the entry process of BTV is similar in specific lipid requirements to enveloped viruses that enter cells through the late endosome. These results are the first, to our knowledge, to demonstrate that a large non-enveloped virus of the Reoviridae has specific lipid requirements for membrane penetration and host cell entry.

Quantification of cooperativity in heterodimer-DNA binding improves the accuracy of binding specificity models [Computational Biology]

February 24th, 2016 by Isakova, A., Berset, Y., Hatzimanikatis, V., Deplancke, B.

Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic PPARγ:RXRα heterodimer. Using the high-throughput MITOMI platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers.

Transcription elongation factor NusA is a general antagonist of Rho-dependent termination in Escherichia coli. [Microbiology]

February 12th, 2016 by Qayyum, M. Z., Dey, D., Sen, R.

NusA is an essential protein that binds to RNA polymerase (RNAP) and also to the nascent RNA, and influences transcription by inducing pausing and facilitating the process of transcription termination / antitermination. Its participation in Rho-dependent transcription termination has been perceived, but the molecular nature of this involvement is not known. We hypothesized that as both Rho and NusA are RNA-binding proteins and have the potential to target the same RNA, the latter is likely to influence the global pattern of the Rho-dependent termination. Analyses of the nascent RNA-binding properties and consequent effects on the Rho-dependent termination functions of specific NusA-RNA binding domain mutants revealed an existence of Rho-NusA direct competition for the overlapping nut (NusA-binding site) and rut (Rho-binding site) sites on the RNA. This leads to delayed entry of Rho at the rut site that inhibits the RNA release process of the latter. High density tiling micro-array profiles of these NusA mutants revealed that a significant number of genes, together with transcripts from intergenic regions are up-regulated. Interestingly, majority of these genes were also up-regulated when the Rho function was compromised. These are strong evidences for the existence of NusA-binding sites in different operons which are also the targets of Rho-dependent terminations. Our data strongly argue in favor of a direct competition between NusA and Rho for the access of specific sites on the nascent transcripts in different parts of the genome. We propose that this competition enables NusA to function as a global antagonist of the Rho function, which is unlike its role as a facilitator of hairpin-dependent termination.

Regulation of Monocarboxylic Acid Transporter 1 Trafficking by the Canonical Wnt/{beta}-catenin Pathway in Rat Brain Endothelial Cells, Requiring a Crosstalk with Notch Signaling [Signal Transduction]

February 12th, 2016 by Liu, Z., Sneve, M., Haroldson, T. A., Smith, J. P., Drewes, L. R.

The transport of monocarboxylate fuels, such as lactate, pyruvate and ketone bodies, across brain endothelial cells is mediated by monocarboxylic acid transporter 1 (MCT1). Although the canonical Wnt/β-catenin pathway is required for rodent blood-brain barrier (BBB) development and for the expression of associated nutrient transporters, the role of this pathway in regulation of brain endothelial MCT1 is unknown. Here, we report expression of nine members of the frizzled receptor family by the RBE4 rat brain endothelial cell line. Furthermore, activation of the canonical Wnt/β-catenin pathway in RBE4 cells via nuclear β-catenin signaling with lithium chloride (LiCl) does not alter brain endothelial Mct1 mRNA, but increases the amount of MCT1 transporter protein. Plasma membrane biotinylation studies and confocal microscopic examination of mCherry-tagged MCT1 indicate that increased transporter results from reduced MCT1 trafficking from the plasma membrane via the endosomal/lysosomal pathway and is facilitated by decreased MCT1 ubiquitination following LiCl treatment. Inhibition of the Notch pathway by the γ-secretase inhibitor, DAPT, negated the upregulation of MCT1 by LiCl, thus demonstrating a crosstalk between the canonical Wnt/β-catenin and Notch pathways. Our results are important because they show for the first time the regulation of MCT1 in cerebrovascular endothelial cells by the multi-functional canonical Wnt/β-catenin and Notch signaling pathways.
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Role of Intrinsic Protein Disorder in the Function and Interactions of the Transcriptional Coactivators CREB-Binding Protein (CBP) and p300 [Signal Transduction]

February 5th, 2016 by Dyson, H. J., Wright, P. E.

The transcriptional coactivators CBP and p300 undergo a particularly rich set of interactions with disordered and partly-ordered partners, as a part of their ubiquitous role in facilitating transcription of genes. CBP and p300 contain a number of small structured domains that provide scaffolds for the interaction of disordered transactivation domains from a wide variety of partners including p53, HIF-1&alpha, NF-&kappaB and STAT proteins, and are the targets for the interactions of disordered viral proteins that compete with cellular factors to disrupt signaling and subvert the cell cycle. The functional diversity of the CBP/p300 interactome provides an excellent example of the power of intrinsic disorder to facilitate the complexity of living systems.
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Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-Dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation [RNA]

February 5th, 2016 by Li, J., Johnson, K. A.

Replication of the Hepatitis C viral genome is catalyzed by the NS5B RNA-dependent RNA polymerase catalyzes, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examine the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates 3-5 nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the β-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information.
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