[Show abstract][Hide abstract] ABSTRACT: Stu2p/XMAP215 proteins are essential microtubule polymerases that use multiple αβ-tubulin-interacting TOG domains to bind microtubule plus ends and catalyze fast microtubule growth. We report here the structure of the TOG2 domain from Stu2p bound to yeast αβ-tubulin. Like TOG1, TOG2 binds selectively to a fully 'curved' conformation of αβ-tubulin, incompatible with a microtubule lattice. We also show that TOG1-TOG2 binds non-cooperatively to two αβ-tubulins. Preferential interactions between TOGs and fully curved αβ-tubulin that cannot exist elsewhere in the microtubule explain how these polymerases localize to the extreme microtubule end. We propose that these polymerases promote elongation because their linked TOG domains concentrate unpolymerized αβ-tubulin near curved subunits already bound at the microtubule end. This tethering model can explain catalyst-like behavior and also predicts that the polymerase action changes the configuration of the microtubule end.
[Show abstract][Hide abstract] ABSTRACT: Colonization of the human nasopharynx by Moraxella catarrhalis is presumed to involve attachment of this bacterium to the mucosa. DNA microarray analysis was used to determine whether attachment of M. catarrhalis to human bronchial epithelial (HBE) cells in vitro affected gene expression in this bacterium. Attachment affected expression of at least 454 different genes, with 163 being up-regulated and 291 being down-regulated. Among the up-regulated genes was one (ORF 113) previously annotated as encoding a protein with some similarity to outer membrane protein A (OmpA). The protein encoded by ORF 113 was predicted to have a signal peptidase II cleavage site, and globomycin inhibition experiments confirmed that this protein was indeed a lipoprotein. The ORF 113 protein also contained a predicted peptidoglycan-binding domain in its C-terminal half. The use of mutant and recombinant M. catarrhalis strains confirmed that the ORF 113 protein was present in outer membrane preparations, and this protein was also shown to be at least partially exposed on the bacterial cell surface. A mutant unable to produce the ORF 113 protein showed little or no change in its growth rate in vitro, in its ability to attach to HBE cells in vitro, or in its autoagglutination characteristics, but did exhibit a reduced ability to survive in the chinchilla nasopharynx. This is the first report of a lipoprotein essential to the ability of M. catarrhalis to persist in an animal model.
Infection and immunity 03/2014; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The alternative sigma factor RpoS in Borrelia burgdorferi plays a central role in modulating host adaptive responses when spirochetes cycle between ticks and mammals. The transcriptional activation of σ54-dependent rpoS requires a Fur homologue designated as BosR. Previously, we found that BosR directly activates rpoS transcription by binding to the rpoS promoter. However, many other DNA-binding features of BosR have remained obscure. In particular, the precise DNA sequence targeted by BosR has not yet been completely elucidated. The prediction of a putative Per box within the rpoS promoter region has further confounded the identification of the BosR binding sequence. Herein, by using electrophoretic mobility shift assays, we demonstrate that the putative Per box predicted in the rpoS promoter region is not involved in the binding of BosR. Rather, a 13-bp palindromic sequence (ATTTAANTTAAAT) with dyad symmetry, which we herein denote as the "BosR box", functions as the core sequence recognized by BosR in the rpoS promoter region of B. burgdorferi. Similar to a Fur box and a Per box, the BosR box likely comprises a 6-1-6 inverted repeat composed of two hexamers (ATTTAA) in a head-to-tail orientation. Selected mutations in the BosR box prevented recombinant BosR from binding to rpoS. In addition, we found that sequences neighboring the BosR box also are required for the formation of BosR-DNA complexes. Identification of the BosR box advances our understanding of how BosR recognizes its DNA target(s), and provides new insight into the mechanistic details behind the unique regulatory function of BosR.
[Show abstract][Hide abstract] ABSTRACT: The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP) synthase (cGAS), which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type I interferons and other cytokines. Here, we report the crystal structures of human cGAS in its apo form, representing its autoinhibited conformation as well as in its cGAMP- and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide insights into the mechanism of DNA sensing by cGAS.
[Show abstract][Hide abstract] ABSTRACT: Accurate measurements of rotor temperature are critical for the interpretation of hydrodynamic parameters in analytical ultracentrifugation. We have recently developed methods for a more accurate determination of the temperature of a spinning rotor utilizing iButton® temperature loggers. Here we report that the temperature measured with the iButton on the counterbalance of a resting rotor, following thermal equilibration under high vacuum, closely corresponded to the temperature of the spinning rotor with a precision better than 0.2 °C. This strategy offers an inexpensive and straightforward approach to monitor the accuracy of the temperature calibration and determine corrective temperature offsets.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT To adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes of Haemophilus ducreyi, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog of hfq. Insertional inactivation of hfq altered the expression of ~16% of the H. ducreyi genes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in the hfq inactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation of hfq downregulated genes in the flp-tad and lspB-lspA2 operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarked hfq deletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion of hfq downregulated Flp1 and impaired the ability of H. ducreyi to form microcolonies, downregulated DsrA and rendered H. ducreyi serum susceptible, and downregulated LspB and LspA2, which allow H. ducreyi to resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor of H. ducreyi stationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog. IMPORTANCE Pathogenic bacteria encounter a wide range of stresses in their hosts, including nutrient limitation; the ability to sense and respond to such stresses is crucial for bacterial pathogens to successfully establish an infection. Gram-negative bacteria frequently utilize the alternative sigma factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, including Haemophilus ducreyi, which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required for H. ducreyi to infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.
[Show abstract][Hide abstract] ABSTRACT: The Lyme disease agent Borrelia burgdorferi, which is transmitted via a tick vector, is dependent on its tick and mammalian hosts for a number of essential nutrients. Like other bacterial diderms, it must transport these biochemicals from the extracellular milieu across two membranes, ultimately to the B. burgdorferi cytoplasm. In the current study, we established that a gene cluster comprising genes bb0215 through bb0218 is cotranscribed and is therefore an operon. Sequence analysis of these proteins suggested that they are the components of an ABC-type transporter responsible for translocating phosphate anions from the B. burgdorferi periplasm to the cytoplasm. Biophysical experiments established that the putative ligand-binding protein of this system, BbPstS (BB0215), binds to phosphate in solution. We determined the high-resolution (1.3 Å) crystal structure of the protein in the absence of phosphate, revealing that the protein's fold is similar to other phosphate-binding proteins, and residues that are implicated in phosphate binding in other such proteins are conserved in BbPstS. Taken together, the gene products of bb0215-0218 function as a phosphate transporter for B. burgdorferi.
[Show abstract][Hide abstract] ABSTRACT: Expression of the lspB-lspA2 operon encoding a virulence-related two-partner secretion system in Haemophilus ducreyi 35000HP is directly regulated by the CpxRA regulatory system [Labandeira-Rey M, Mock JR, Hansen E. Regulation of expression of the Haemophilus ducreyi LspB and LspA2 proteins by CpxR. Infect. Immun. 77: 3402-3411 (2009)]. In the present study, we show that this secretion system is also regulated by the small nucleoid-associated protein Fis. Inactivation of the H. ducreyi fis gene resulted in a reduction in expression of both the H. ducreyi LspB and LspA2 proteins. DNA microarray experiments showed that a H. ducreyi fis deletion mutant exhibited altered expression of genes encoding other important H. ducreyi virulence factors including DsrA and Flp1, suggesting a possible global role for Fis in control of virulence in this obligate human pathogen. While the H. ducreyi Fis protein has a high degree of sequence and structural similarity to the Fis proteins of other bacteria, its temporal pattern of expression was very different from that of enterobacterial Fis proteins. The use of a lacZ-based transcriptional reporter provided evidence which indicated that the H. ducreyi Fis homolog is a positive regulator of gyrB, a gene that is negatively regulated by Fis in enteric bacteria. Taken together, the Fis protein expression data and the observed regulatory effects of Fis in H. ducreyi suggest that this small DNA binding protein has a regulatory role in H. ducreyi which may differ in substantial ways from that of other Fis proteins.
Infection and immunity 08/2013; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tumor-suppressive Hippo pathway controls tissue homeostasis through balancing cell proliferation and apoptosis. Activation of the kinases Mst1 and Mst2 (Mst1/2) is a key upstream event in this pathway and remains poorly understood. Mst1/2 and their critical regulators RASSFs contain Salvador/RASSF1A/Hippo (SARAH) domains that can homo- and heterodimerize. Here, we report the crystal structures of human Mst2 alone and bound to RASSF5. Mst2 undergoes activation through transautophosphorylation at its activation loop, which requires SARAH-mediated homodimerization. RASSF5 disrupts Mst2 homodimer and blocks Mst2 autoactivation. Binding of RASSF5 to already activated Mst2, however, does not inhibit its kinase activity. Thus, RASSF5 can act as an inhibitor or a potential positive regulator of Mst2, depending on whether it binds to Mst2 before or after activation-loop phosphorylation. We propose that these temporally sensitive functions of RASSFs enable the Hippo pathway to respond to and integrate diverse cellular signals.
[Show abstract][Hide abstract] ABSTRACT: Cohesin, along with positive regulators, establishes sister-chromatid cohesion by forming a ring to circle chromatin. The wings apart-like protein (Wapl) is a key negative regulator of cohesin and forms a complex with precocious dissociation of sisters protein 5 (Pds5) to promote cohesin release from chromatin. Here we report the crystal structure and functional characterization of human Wapl. Wapl contains a flexible, variable N-terminal region (Wapl-N) and a conserved C-terminal domain (Wapl-C) consisting of eight HEAT (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) repeats. Wapl-C folds into an elongated structure with two lobes. Structure-based mutagenesis maps the functional surface of Wapl-C to two distinct patches (I and II) on the N lobe and a localized patch (III) on the C lobe. Mutating critical patch I residues weaken Wapl binding to cohesin and diminish sister-chromatid resolution and cohesin release from mitotic chromosomes in human cells and Xenopus egg extracts. Surprisingly, patch III on the C lobe does not contribute to Wapl binding to cohesin or its known regulators. Although patch I mutations reduce Wapl binding to intact cohesin, they do not affect Wapl-Pds5 binding to the cohesin subcomplex of sister chromatid cohesion protein 1 (Scc1) and stromal antigen 2 (SA2) in vitro, which is instead mediated by Wapl-N. Thus, Wapl-N forms extensive interactions with Pds5 and Scc1-SA2. Wapl-C interacts with other cohesin subunits and possibly unknown effectors to trigger cohesin release from chromatin.
Proceedings of the National Academy of Sciences 06/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sedimentation velocity (SV) is a method based on first-principles that provides a precise hydrodynamic characterization of macromolecules in solution. Due to recent improvements in data analysis, the accuracy of experimental SV data emerges as a limiting factor in its interpretation. Our goal was to unravel the sources of experimental error and develop improved calibration procedures. We implemented the use of a Thermochron iButton® temperature logger to directly measure the temperature of a spinning rotor, and detected deviations that can translate into an error of as much as 10% in the sedimentation coefficient. We further designed a precision mask with equidistant markers to correct for instrumental errors in the radial calibration, which were observed to span a range of 8.6%. The need for an independent time calibration emerged with use of the current data acquisition software (Zhao et al., doi 10.1016/j.ab.2013.02.011) and we now show that smaller but significant time errors of up to 2% also occur with earlier versions. After application of these calibration corrections, the sedimentation coefficients obtained from eleven instruments displayed a significantly reduced standard deviation of ∼ 0.7%. This study demonstrates the need for external calibration procedures and regular control experiments with a sedimentation coefficient standard.
[Show abstract][Hide abstract] ABSTRACT: Syphilis, caused by the bacterial spirochete Treponema pallidum, remains a prominent sexually transmitted infection worldwide. Despite sequencing of the genome of this obligate human pathogen 15 years ago, the functions of a large number of the gene products of T. pallidum are still unknown, particularly with respect to those of the organism's periplasmic lipoproteins. To better understand their functions, a structural biology approach has been pursued. To this end, the soluble portion of the T. pallidum TP0435 lipoprotein (also known as Tp17) was cloned, hyper-expressed in Escherichia coli and purified to apparent homogeneity. The protein crystals obtained from this preparation diffracted to 2.4 Å resolution and had the symmetry of space group R3. In the hexagonal setting, the unit-cell parameters were a = b = 85.7, c = 85.4 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2013; 69(Pt 4):453-5. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report systematic and large inaccuracies in the recorded elapsed time in data files from the analytical ultracentrifuge, leading to overestimates of the sedimentation coefficients of up to 10%. This far exceeds previously considered factors contributing to the uncertainty in this parameter, and has significant ramifications for derived parameters, such as hydrodynamic shape and molar mass estimates. The source of this error is at present unknown, but we found it to be quantitatively consistent across different instruments, increasing with rotor speed. Furthermore, its occurrence appears to correlate with the use of the latest data acquisition software from the manufacturer, in use in some of our laboratories for nearly two years. Many of the recently published sedimentation coefficients may need to be re-examined. The problem can be easily recognized by comparing the file time-stamps provided by the operating system with the elapsed scan times recorded within the data files. We therefore implemented a routine in SEDFIT that can automatically examine the data files, alert the user to significant discrepancies, and correct the scan times accordingly. This eliminates errors in the recorded scan times.
[Show abstract][Hide abstract] ABSTRACT: Treponema pallidum (Tp), an obligate parasite of humans and the causative agent of syphilis, has evolved the capacity to exploit host-derived metabolites for its survival. Flavin-containing compounds are essential cofactors that are required for metabolic processes in all living organisms, and riboflavin is a direct precursor of the cofactors FMN and FAD. Unlike many pathogenic bacteria, Tp cannot synthesize riboflavin; we recently described a flavin-uptake mechanism comprised of an ABC-type transporter. However, there is a paucity of information about flavin utilization in bacterial periplasms. Using a discovery-driven approach, we have identified the TP0796 lipoprotein as a previously uncharacterized Mg2+-dependent FAD pyrophosphatase within the ApbE superfamily. TP0796 likely plays a central role in flavin turnover by hydrolyzing exogenously acquired FAD yielding AMP and FMN. Biochemical and structural investigations revealed that the enzyme has a unique bimetal Mg2+catalytic center. Furthermore, the pyrophosphatase activity is product inhibited by AMP, indicating a possible role for this molecule in modulating FMN and FAD levels in the treponemal periplasm. The ApbE superfamily was previously thought to be involved in thiamine biosynthesis, but our characterization of TP0796 prompts a re-naming of this superfamily as a periplasmic FAD processing protein (Fpp). TP0796 is the first structurally and biochemically characterized FAD pyrophosphate enzyme in bacteria. This new paradigm for a bacterial flavin-utilization pathway may prove to be useful for future inhibitor design.
Journal of Biological Chemistry 02/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human African trypanosomiasis (HAT) is caused by a single-celled protozoan parasite, Trypanosoma brucei. Polyamine biosynthesis is a clinically validated target for the treatment of HAT. Metabolic differences between the parasite and the human polyamine pathway are thought to contribute to species selectivity of pathway inhibitors. S-adenosylmethionine decarboxylase (AdoMetDC) catalyses a key step in the production of the polyamine spermidine. We previously showed that trypanosomatid AdoMetDC differs from other eukaryotic enzymes in that it is regulated by heterodimer formation with a catalytically dead paralog, designated prozyme, which binds with high affinity to the enzyme and increases its activity by up to 103-fold. Herein, we examine the role of specific residues involved in AdoMetDC activation by prozyme through deletion and site-directed mutagenesis. Results indicate that 12 key amino acids at the N-terminus of AdoMetDC are essential for prozyme-mediated activation, with Leu-8, Leu-10, Met-11 and Met-13 identified as the key residues. These N-terminal residues are fully conserved in the trypanosomatids but are absent from other eukaryotic homologs lacking the prozyme mechanism, suggesting co-evolution of these residues with the prozyme mechanism. Heterodimer formation between AdoMetDC and prozyme was not impaired by mutation of Leu-8 and Leu-10 to Ala, suggesting that these residues are involved in a conformational change that is essential for activation. Our findings provide the first insight into the mechanisms that influence catalytic regulation of AdoMetDC, and may have potential implications for the development of new inhibitors against this enzyme.
Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plexins are cell surface receptors that bind semaphorins and transduce signals for regulating neuronal axon guidance and other processes. Plexin signaling depends on their cytoplasmic GTPase activating protein (GAP) domain, which specifically inactivates the Ras homolog Rap through an ill-defined non-canonical catalytic mechanism. The plexin GAP is activated by semaphorin-induced dimerization, the structural basis for which remained unknown. Here we present the crystal structures of the active dimer of zebrafish PlexinC1 cytoplasmic region in the apo state and in complex with Rap. The structures show that the dimerization induces a large-scale conformational change in plexin, which opens the GAP active site to allow Rap binding. Plexin stabilizes the switch II region of Rap in an unprecedented conformation, bringing Gln63 in Rap into the active site for catalyzing GTP hydrolysis. The structures also explain the unique Rap-specificity of plexins. Mutational analyses support that these mechanisms underlie plexin activation and signaling. DOI:http://dx.doi.org/10.7554/eLife.01279.001.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Bacterial transporter proteins are involved in the translocation of many essential nutrients and metabolites. However, many of these key bacterial transport systems remain to be identified, including those involved in the transport of riboflavin (vitamin B(2)). Pathogenic spirochetes lack riboflavin biosynthetic pathways, implying reliance on obtaining riboflavin from their hosts. Using structural and functional characterizations of possible ligand-binding components, we have identified an ABC-type riboflavin transport system within pathogenic spirochetes. The putative lipoprotein ligand-binding components of these systems from three different spirochetes were cloned, hyperexpressed in Escherichia coli, and purified to homogeneity. Solutions of all three of the purified recombinant proteins were bright yellow. UV-visible spectra demonstrated that these proteins were likely flavoproteins; electrospray ionization mass spectrometry and thin-layer chromatography confirmed that they contained riboflavin. A 1.3-Å crystal structure of the protein (TP0298) encoded by Treponema pallidum, the syphilis spirochete, demonstrated that the protein's fold is similar to the ligand-binding components of ABC-type transporters. The structure also revealed other salient details of the riboflavin binding site. Comparative bioinformatics analyses of spirochetal genomes, coupled with experimental validation, facilitated the discovery of this new ABC-type riboflavin transport system(s). We denote the ligand-binding component as riboflavin uptake transporter A (RfuA). Taken together, it appears that pathogenic spirochetes have evolved an ABC-type transport system (RfuABCD) for survival in their host environments, particularly that of the human host. IMPORTANCE Syphilis remains a public health problem, but very little is known about the causative bacterium. This is because Treponema pallidum still cannot be cultured in the laboratory. Rather, T. pallidum must be cultivated in laboratory rabbits, a restriction that poses many insurmountable experimental obstacles. Approaches to learn more about the structure and function of T. pallidum's cell envelope, which is both the physical and functional interface between T. pallidum and its human host, are severely limited. One approach for elucidating T. pallidum's cell envelope has been to determine the three-dimensional structures of its membrane lipoproteins, molecules that serve many critical survival functions. Herein, we describe a previously unknown transport system that T. pallidum uses to import riboflavin, an essential nutrient for the organism's survival. Moreover, we found that this transport system is present in other pathogenic spirochetes. This is the first description of this new type of bacterial riboflavin transport system.
[Show abstract][Hide abstract] ABSTRACT: Multi-signal sedimentation velocity analytical ultracentrifugation (MSSV) is a powerful tool for the determination of the number, stoichiometry, and hydrodynamic shape of reversible protein complexes in two- and three-component systems. In this method, the evolution of sedimentation profiles of macromolecular mixtures is recorded simultaneously using multiple absorbance and refractive index signals and globally transformed into both spectrally and diffusion-deconvoluted component sedimentation coefficient distributions. For reactions with complex lifetimes comparable to the time-scale of sedimentation, MSSV reveals the number and stoichiometry of co-existing complexes. For systems with short complex lifetimes, MSSV reveals the composition of the reaction boundary of the coupled reaction/migration process, which we show here may be used to directly determine an association constant. A prerequisite for MSSV is that the interacting components are spectrally distinguishable, which may be a result, for example, of extrinsic chromophores or of different abundances of aromatic amino acids contributing to the UV absorbance. For interacting components that are spectrally poorly resolved, here we introduce a method for additional regularization of the spectral deconvolution by exploiting approximate knowledge of the total loading concentrations. While this novel mass conservation principle does not discriminate contributions to different species, it can be effectively combined with constraints in the sedimentation coefficient range of uncomplexed species. We show in theory, computer simulations, and experiment, how mass conservation MSSV as implemented in SEDPHAT can enhance or even substitute for the spectral discrimination of components. This should broaden the applicability of MSSV to the analysis of the composition of reversible macromolecular complexes.
PLoS ONE 01/2013; 8(5):e62694. · 3.73 Impact Factor