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Sample records for conserved residue clusters

  1. Distantly related lipocalins share two conserved clusters of hydrophobic residues: use in homology modeling

    PubMed Central

    Adam, Benoit; Charloteaux, Benoit; Beaufays, Jerome; Vanhamme, Luc; Godfroid, Edmond; Brasseur, Robert; Lins, Laurence

    2008-01-01

    Background Lipocalins are widely distributed in nature and are found in bacteria, plants, arthropoda and vertebra. In hematophagous arthropods, they are implicated in the successful accomplishment of the blood meal, interfering with platelet aggregation, blood coagulation and inflammation and in the transmission of disease parasites such as Trypanosoma cruzi and Borrelia burgdorferi. The pairwise sequence identity is low among this family, often below 30%, despite a well conserved tertiary structure. Under the 30% identity threshold, alignment methods do not correctly assign and align proteins. The only safe way to assign a sequence to that family is by experimental determination. However, these procedures are long and costly and cannot always be applied. A way to circumvent the experimental approach is sequence and structure analyze. To further help in that task, the residues implicated in the stabilisation of the lipocalin fold were determined. This was done by analyzing the conserved interactions for ten lipocalins having a maximum pairwise identity of 28% and various functions. Results It was determined that two hydrophobic clusters of residues are conserved by analysing the ten lipocalin structures and sequences. One cluster is internal to the barrel, involving all strands and the 310 helix. The other is external, involving four strands and the helix lying parallel to the barrel surface. These clusters are also present in RaHBP2, a unusual "outlier" lipocalin from tick Rhipicephalus appendiculatus. This information was used to assess assignment of LIR2 a protein from Ixodes ricinus and to build a 3D model that helps to predict function. FTIR data support the lipocalin fold for this protein. Conclusion By sequence and structural analyzes, two conserved clusters of hydrophobic residues in interactions have been identified in lipocalins. Since the residues implicated are not conserved for function, they should provide the minimal subset necessary to confer the

  2. The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2*

    PubMed Central

    Chen, Xinhuan; Xu, Zhijian; Zhang, Lingna; Liu, Hongchuan; Liu, Xia; Lou, Meng; Zhu, Lijun; Huang, Bingding; Yang, Cai-Guang; Zhu, Weiliang; Shao, Jimin

    2014-01-01

    Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G1/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells. PMID:24253041

  3. A Highly Conserved Residue of the HIV-1 gp120 Inner Domain Is Important for Antibody-Dependent Cellular Cytotoxicity Responses Mediated by Anti-cluster A Antibodies

    PubMed Central

    Ding, Shilei; Veillette, Maxime; Coutu, Mathieu; Prévost, Jérémie; Scharf, Louise; Bjorkman, Pamela J.; Ferrari, Guido; Robinson, James E.; Stürzel, Christina; Hahn, Beatrice H.; Sauter, Daniel; Kirchhoff, Frank; Lewis, George K.; Pazgier, Marzena

    2015-01-01

    Previous studies have shown that sera from HIV-1-infected individuals contain antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC). These antibodies preferentially recognize envelope glycoprotein (Env) epitopes induced upon CD4 binding. Here, we show that a highly conserved tryptophan at position 69 of the gp120 inner domain is important for ADCC mediated by anti-cluster A antibodies and sera from HIV-1-infected individuals. PMID:26637462

  4. Molybdenum cofactor properties and [Fe-S] cluster coordination in Escherichia coli nitrate reductase A: investigation by site-directed mutagenesis of the conserved his-50 residue in the NarG subunit.

    PubMed

    Magalon, A; Asso, M; Guigliarelli, B; Rothery, R A; Bertrand, P; Giordano, G; Blasco, F

    1998-05-19

    Most of the molybdoenzymes contain, in the amino-terminal region of their catalytic subunits, a conserved Cys group that in some cases binds an [Fe-S] cluster. In dissimilatory nitrate reductases, the first Cys residue of this motif is replaced by a conserved His residue. Site-directed mutagenesis of this residue (His-50) was performed on the NarG subunit from Escherichia coli nitrate reductase A. The results obtained by EPR spectroscopy enable us to exclude the implication of this residue in [Fe-S] binding. Additionally, we showed that the His-50 residue does not coordinate the molybdenum atom, but its substitution by Cys or Ser introduces a perturbation of the hydrogen bonding network around the molybdenum cofactor. From potentiometric studies, it is proposed that the high-pH and the low-pH forms of the Mo(V) are both involved during the redox turnover of the enzyme. Perturbation of the Mo(V) pKV value might be responsible for the low activity reported in the His-50-Cys mutant enzyme. A catalytic model is proposed in which the protonation/deprotonation of the Mo(V) species is an essential step. Thus, one of the two protons involved in the catalytic cycle could be the one coupled to the molybdenum atom in the dissimilatory nitrate reductase of E. coli. PMID:9585550

  5. Conservation Level and Category Clustering

    ERIC Educational Resources Information Center

    Haynes, C. Rayfield; Kulhavy, Raymond W.

    1976-01-01

    Two experiments were conducted to test the hypothesis that category recall is related to the quantity conservation of mass, weight, and volume. The predicted association between conservation level and category recall was observed. (JMB)

  6. Conservation value of clustered housing developments.

    PubMed

    Lenth, Buffy A; Knight, Richard L; Gilgert, Wendell C

    2006-10-01

    Traditionally, exurban lands in Colorado have been subdivided into a grid of parcels ranging from 2 to 16 ha. From an ecological perspective, this dispersed pattern of development effectively maximizes the individual influence of each home on the land. Clustered housing developments, designed to maximize open space, are assumed to benefit plant and wildlife communities of conservation interest. They have become a popular alternative for rural development despite the lack of empirical evidence demonstrating their conservation benefits. To better inform rural land-use planning, we evaluated clustered housing developments by comparing their spatial pattern with that of dispersed housing developments and by comparing their conservation value with that of both dispersed housing developments and undeveloped areas in Boulder County, Colorado. We used four indicators to assess conservation value: (1) densities of songbirds, (2) nest density and survival of ground-nesting birds, (3) presence of mammals, and (4) percent cover and proportion of native and non-native plant species. Clustered and dispersed housing developments did not differ on the majority of variables we examined. Both types of housing development had significantly higher densities of non-native and human-commensal species and significantly lower densities of native and human-sensitive species than undeveloped areas. More rigorous ecological guidelines and planning on a regional scale may help create clustered developments with higher conservation value. PMID:17002762

  7. 3. VIEW NORTHEAST, SOUTH FRONT OF SOIL CONSERVATION SERVICE CLUSTER ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. VIEW NORTHEAST, SOUTH FRONT OF SOIL CONSERVATION SERVICE CLUSTER (BUILDING 25) - U.S. Plant Introduction Station, Soil Conservation Service Cluster, 11601 Old Pond Road, Glenn Dale, Prince George's County, MD

  8. 1. VIEW EAST, WEST FRONT OF SOIL CONSERVATION SERVICE CLUSTER ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. VIEW EAST, WEST FRONT OF SOIL CONSERVATION SERVICE CLUSTER (BUILDINGS 24, 25, 26) - U.S. Plant Introduction Station, Soil Conservation Service Cluster, 11601 Old Pond Road, Glenn Dale, Prince George's County, MD

  9. 4. VIEW NORTHWEST, NORTH FRONT OF SOIL CONSERVATION SERVICE CLUSTER ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. VIEW NORTHWEST, NORTH FRONT OF SOIL CONSERVATION SERVICE CLUSTER (BUILDINGS 24, 25, 26); NORTH FRONT OF QUARANTINE HEADHOUSE (BUILDING 27) - U.S. Plant Introduction Station, Soil Conservation Service Cluster, 11601 Old Pond Road, Glenn Dale, Prince George's County, MD

  10. Effective function annotation through catalytic residue conservation.

    PubMed

    George, Richard A; Spriggs, Ruth V; Bartlett, Gail J; Gutteridge, Alex; MacArthur, Malcolm W; Porter, Craig T; Al-Lazikani, Bissan; Thornton, Janet M; Swindells, Mark B

    2005-08-30

    Because of the extreme impact of genome sequencing projects, protein sequences without accompanying experimental data now dominate public databases. Homology searches, by providing an opportunity to transfer functional information between related proteins, have become the de facto way to address this. Although a single, well annotated, close relationship will often facilitate sufficient annotation, this situation is not always the case, particularly if mutations are present in important functional residues. When only distant relationships are available, the transfer of function information is more tenuous, and the likelihood of encountering several well annotated proteins with different functions is increased. The consequence for a researcher is a range of candidate functions with little way of knowing which, if any, are correct. Here, we address the problem directly by introducing a computational approach to accurately identify and segregate related proteins into those with a functional similarity and those where function differs. This approach should find a wide range of applications, including the interpretation of genomics/proteomics data and the prioritization of targets for high-throughput structure determination. The method is generic, but here we concentrate on enzymes and apply high-quality catalytic site data. In addition to providing a series of comprehensive benchmarks to show the overall performance of our approach, we illustrate its utility with specific examples that include the correct identification of haptoglobin as a nonenzymatic relative of trypsin, discrimination of acid-d-amino acid ligases from a much larger ligase pool, and the successful annotation of BioH, a structural genomics target. PMID:16037208

  11. Comparing Residue Clusters from Thermophilic and Mesophilic Enzymes Reveals Adaptive Mechanisms

    PubMed Central

    Sammond, Deanne W.; Kastelowitz, Noah; Himmel, Michael E.; Yin, Hang; Crowley, Michael F.; Bomble, Yannick J.

    2016-01-01

    Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research. Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. Thus the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions. PMID:26741367

  12. Comparing residue clusters from thermophilic and mesophilic enzymes reveals adaptive mechanisms

    DOE PAGESBeta

    Sammond, Deanne W.; Kastelowitz, Noah; Himmel, Michael E.; Yin, Hang; Crowley, Michael F.; Bomble, Yannick J.

    2016-01-07

    Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research.more » Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. As a result, the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions.« less

  13. Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

    SciTech Connect

    Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

    2003-12-31

    Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involved in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.

  14. Residual Distribution Schemes for Conservation Laws Via Adaptive Quadrature

    NASA Technical Reports Server (NTRS)

    Barth, Timothy; Abgrall, Remi; Biegel, Bryan (Technical Monitor)

    2000-01-01

    This paper considers a family of nonconservative numerical discretizations for conservation laws which retains the correct weak solution behavior in the limit of mesh refinement whenever sufficient order numerical quadrature is used. Our analysis of 2-D discretizations in nonconservative form follows the 1-D analysis of Hou and Le Floch. For a specific family of nonconservative discretizations, it is shown under mild assumptions that the error arising from non-conservation is strictly smaller than the discretization error in the scheme. In the limit of mesh refinement under the same assumptions, solutions are shown to satisfy an entropy inequality. Using results from this analysis, a variant of the "N" (Narrow) residual distribution scheme of van der Weide and Deconinck is developed for first-order systems of conservation laws. The modified form of the N-scheme supplants the usual exact single-state mean-value linearization of flux divergence, typically used for the Euler equations of gasdynamics, by an equivalent integral form on simplex interiors. This integral form is then numerically approximated using an adaptive quadrature procedure. This renders the scheme nonconservative in the sense described earlier so that correct weak solutions are still obtained in the limit of mesh refinement. Consequently, we then show that the modified form of the N-scheme can be easily applied to general (non-simplicial) element shapes and general systems of first-order conservation laws equipped with an entropy inequality where exact mean-value linearization of the flux divergence is not readily obtained, e.g. magnetohydrodynamics, the Euler equations with certain forms of chemistry, etc. Numerical examples of subsonic, transonic and supersonic flows containing discontinuities together with multi-level mesh refinement are provided to verify the analysis.

  15. Residue Management: A Computer Program About Conservation Tillage Decisions.

    ERIC Educational Resources Information Center

    Thien, Steve J.

    1986-01-01

    Describes a computer program, Residue Management, which is designed to supplement discussions on the Universal Soil Loss Equation and the impact of tillage on soil properties for introductory soil courses. The program advances the user through three stages of residue management. Information on obtaining the program is also included. (ML)

  16. Herbicide and cover crop residue integration in conservation tillage tomato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increased adoption of conservation tillage in vegetable production requires more information on the role of various cover crops in weed control, tomato quality, and yield. Three conservation-tillage systems utilizing crimson clover, turnip, and cereal rye as winter cover crops were compared to a...

  17. Structural consequences of chromophore formation and exploration of conserved lid residues amongst naturally occurring fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Zimmer, Matthew H.; Li, Binsen; Shahid, Ramza; Peshkepija, Paola; Zimmer, Marc

    2014-01-01

    Computational methods were used to generate the lowest energy conformations of the immature precyclized forms of the 28 naturally occurring GFP-like proteins deposited in the pdb. In all 28 GFP-like proteins, the beta-barrel contracts upon chromophore formation and becomes more rigid. Our prior analysis of over 260 distinct naturally occurring GFP-like proteins revealed that most of the conserved residues are located in the top and bottom of the barrel in the turns between the β-sheets (Ong et al. 2011) [1]. Structural analyses, molecular dynamics simulations and the Anisotropic Network Model were used to explore the role of these conserved lid residues as possible folding nuclei. Our results are internally consistent and show that the conserved residues in the top and bottom lids undergo relatively less translational movement than other lid residues, and a number of these residues may play an important role as hinges or folding nuclei in the fluorescent proteins.

  18. MCMC Sampling for a Multilevel Model with Nonindependent Residuals within and between Cluster Units

    ERIC Educational Resources Information Center

    Browne, William; Goldstein, Harvey

    2010-01-01

    In this article, we discuss the effect of removing the independence assumptions between the residuals in two-level random effect models. We first consider removing the independence between the Level 2 residuals and instead assume that the vector of all residuals at the cluster level follows a general multivariate normal distribution. We…

  19. A conserved activation cluster is required for allosteric communication in HtrA-family proteases.

    PubMed

    de Regt, Anna K; Kim, Seokhee; Sohn, Jungsan; Grant, Robert A; Baker, Tania A; Sauer, Robert T

    2015-03-01

    In E. coli, outer-membrane stress causes a transcriptional response through a signaling cascade initiated by DegS cleavage of a transmembrane antisigma factor. Each subunit of DegS, an HtrA-family protease, contains a protease domain and a PDZ domain. The trimeric protease domain is autoinhibited by the unliganded PDZ domains. Allosteric activation requires binding of unassembled outer-membrane proteins (OMPs) to the PDZ domains and protein substrate binding. Here, we identify a set of DegS residues that cluster together at subunit-subunit interfaces in the trimer, link the active sites and substrate binding sites, and are crucial for stabilizing the active enzyme conformation in response to OMP signaling. These residues are conserved across the HtrA-protease family, including orthologs linked to human disease, supporting a common mechanism of allosteric activation. Indeed, mutation of residues at homologous positions in the DegP quality-control protease also eliminates allosteric activation. PMID:25703375

  20. Impact of interfacial tension on residual CO2 clusters in porous sandstone

    NASA Astrophysics Data System (ADS)

    Jiang, Fei; Tsuji, Takeshi

    2015-03-01

    We develop a numerical simulation that uses the lattice Boltzmann method to directly calculate the characteristics of residual nonwetting-phase clusters to quantify capillary trapping mechanisms in real sandstone. For this purpose, a digital-rock-pore model reconstructed from micro-CT-scanned images of Berea sandstone is filtered and segmented into a binary file. The residual-cluster distribution is generated following simulation of the drainage and imbibition processes. The characteristics of the residual cluster in terms of size distribution, major length, interfacial area, and sphericity are investigated under conditions of different interfacial tension (IFT). Our results indicate that high interfacial tension increases the residual saturation and leads to a large size distribution of residual clusters. However, low interfacial tension results in a larger interfacial area, which is beneficial for dissolution and reaction processes during geological carbon storage. Analysis of the force balance acting on the residual clusters demonstrates that trapping stability is higher in high interfacial tension case, and the interfacial tension should be a controlling factor for the trapping stability in addition to the pore geometry and connectivity. The proposed numerical method can handle the complex displacement of multicomponent systems in porous media. By using this method, we can obtain residual-cluster distributions under different conditions for optimizing the storage capacity of carbon-storage projects.

  1. Estimating residue evolutionary conservation by introducing von Neumann entropy and a novel gap-treating approach.

    PubMed

    Zhang, S-W; Zhang, Y-L; Pan, Q; Cheng, Y-M; Chou, K-C

    2008-08-01

    Evolutionary conservation derived from a multiple sequence alignment is a powerful indicator of the functional significance of a residue, and it can help to predict active sites, ligand-binding sites, and protein interaction interfaces. The results of the existing algorithms in identifying the residue's conservation strongly depend on the sequence alignment, making the results highly variable. Here, by introducing the amino acid similarity matrix, we propose a novel gap-treating approach by combining the evolutionary information and von Neumann entropies to compute the residue conservation scores. It is indicated through a series of tested results that the new approach is quite encouraging and promising and may become a useful tool in complementing the existing methods. PMID:17710364

  2. Functional analysis of conserved residues in the putative "finger" domain of telomerase reverse transcriptase.

    PubMed

    Bosoy, D; Lue, N F

    2001-12-01

    Telomerase is a ribonucleoprotein reverse transcriptase (RT) responsible for the maintenance of one strand of telomere terminal repeats. The catalytic protein subunit of telomerase, known generically as telomerase reverse transcriptase (TERT), exhibits significant homology to RTs encoded by retroviruses and retroelements. The polymerization mechanisms of telomerase may therefore be similar to those of the "conventional" RTs. In this study, we explored the extent of mechanistic conservation by analyzing mutations of conserved residues within the putative "finger" domain of TERT. Previous analysis has implicated this domain of retroviral RTs in nucleotide and RNA binding and in processivity control. Our results demonstrate that residues conserved between TERT and human immunodeficiency virus-1 RT are more likely than TERT-specific residues to be required for enzyme activity. In addition, residues presumed to make direct contact with either the RNA or nucleotide substrate appear to be functionally more important. Furthermore, distinct biochemical defects can be observed for alterations in the putative RNA- and nucleotide-binding TERT residues in a manner that can be rationalized by their postulated mechanisms of action. This study thus supports a high degree of mechanistic conservation between telomerase and retroviral RTs and underscores the roles of distinct aspects of telomerase biochemistry in telomere length maintenance. PMID:11581271

  3. The role of a conserved acidic residue in calcium-dependent protein folding for a low density lipoprotein (LDL)-A module: implications in structure and function for the LDL receptor superfamily.

    PubMed

    Guo, Ying; Yu, Xuemei; Rihani, Kayla; Wang, Qing-Yin; Rong, Lijun

    2004-04-16

    One common feature of the more than 1,000 complement-type repeats (or low density lipoprotein (LDL)-A modules) found in LDL receptor and the other members of the LDL receptor superfamily is a cluster of five highly conserved acidic residues in the C-terminal region, DXXXDXXDXXDE. However, the role of the third conserved aspartate of these LDL-A modules in protein folding and ligand recognition has not been elucidated. In this report, using a model LDL-A module and several experimental approaches, we demonstrate that this acidic residue, like the other four conserved acidic residues, is involved in calcium-dependent protein folding. These results suggest an alternative calcium coordination conformation for the LDL-A modules. The proposed model provides a plausible explanation for the conservation of this acidic residue among the LDL-A modules. Furthermore, the model can explain why mutations of this residue in human LDL receptor cause familial hypercholesterolemia. PMID:14749324

  4. Recent Galaxy Mergers and Residual Star Formation of Red Sequence Galaxies in Galaxy Clusters

    NASA Astrophysics Data System (ADS)

    Sheen, Yun-Kyeong; Yi, Sukyoung K.; Ree, Chang H.; Jaffé, Yara; Demarco, Ricardo; Treister, Ezequiel

    2016-08-01

    This study explored the Galaxy Evolution Explorer ultraviolet (UV) properties of optical red sequence galaxies in four rich Abell clusters at z≤slant 0.1. In particular, we tried to find a hint of merger-induced recent star formation (RSF) in red sequence galaxies. Using the NUV - r\\prime colors of the galaxies, RSF fractions were derived based on various criteria for post-merger galaxies and normal galaxies. Following k-correction, about 36% of the post-merger galaxies were classified as RSF galaxies with a conservative criterion (NUV - r\\prime ≤slant 5), and that number was doubled (∼72%) when using a generous criterion (NUV - r\\prime ≤slant 5.4). The trend was the same when we restricted the sample to galaxies within 0.5 × R 200. Post-merger galaxies with strong UV emission showed more violent, asymmetric features in the deep optical images. The RSF fractions did not show any trend along the clustocentric distance within R 200. We performed a Dressler–Shectman test to check whether the RSF galaxies had any correlation with the substructures in the galaxy clusters. Within R 200 of each cluster, the RSF galaxies did not appear to be preferentially related to the clusters’ substructures. Our results suggested that only 30% of RSF red sequence galaxies show morphological hints of recent galaxy mergers. This implies that internal processes (e.g., stellar mass loss or hot gas cooling) for the supply of cold gas to early-type galaxies may play a significant role in the residual star formation of early-type galaxies at a recent epoch.

  5. Recent Galaxy Mergers and Residual Star Formation of Red Sequence Galaxies in Galaxy Clusters

    NASA Astrophysics Data System (ADS)

    Sheen, Yun-Kyeong; Yi, Sukyoung K.; Ree, Chang H.; Jaffé, Yara; Demarco, Ricardo; Treister, Ezequiel

    2016-08-01

    This study explored the Galaxy Evolution Explorer ultraviolet (UV) properties of optical red sequence galaxies in four rich Abell clusters at z≤slant 0.1. In particular, we tried to find a hint of merger-induced recent star formation (RSF) in red sequence galaxies. Using the NUV - r\\prime colors of the galaxies, RSF fractions were derived based on various criteria for post-merger galaxies and normal galaxies. Following k-correction, about 36% of the post-merger galaxies were classified as RSF galaxies with a conservative criterion (NUV - r\\prime ≤slant 5), and that number was doubled (˜72%) when using a generous criterion (NUV - r\\prime ≤slant 5.4). The trend was the same when we restricted the sample to galaxies within 0.5 × R 200. Post-merger galaxies with strong UV emission showed more violent, asymmetric features in the deep optical images. The RSF fractions did not show any trend along the clustocentric distance within R 200. We performed a Dressler–Shectman test to check whether the RSF galaxies had any correlation with the substructures in the galaxy clusters. Within R 200 of each cluster, the RSF galaxies did not appear to be preferentially related to the clusters’ substructures. Our results suggested that only 30% of RSF red sequence galaxies show morphological hints of recent galaxy mergers. This implies that internal processes (e.g., stellar mass loss or hot gas cooling) for the supply of cold gas to early-type galaxies may play a significant role in the residual star formation of early-type galaxies at a recent epoch.

  6. Herbicide and cover crop residue integration for amaranth control in conservation agriculture cotton and implications for resistance management

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conservation agriculture practices are threatened by glyphosate-resistant Palmer amaranth. Integrated practices including PRE herbicides and high-residue conservation agriculture systems may decrease Amaranth emergence. Field experiments were conducted from autumn 2006 through cash crop harvest in...

  7. Site-directed mutagenesis of conserved cysteine residues in NqrD and NqrE subunits of Na+-translocating NADH:quinone oxidoreductase.

    PubMed

    Fadeeva, M S; Bertsova, Y V; Verkhovsky, M I; Bogachev, A V

    2008-02-01

    Each of two hydrophobic subunits of Na+-translocating NADH:quinone oxidoreductase (NQR), NqrD and NqrE, contain a pair of strictly conserved cysteine residues within their transmembrane alpha-helices. Site-directed mutagenesis showed that substitutions of these residues in NQR of Vibrio harveyi blocked the Na+-dependent and 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive quinone reductase activity of the enzyme. However, these mutations did not affect the interaction of NQR with NADH and menadione. It was demonstrated that these conserved cysteine residues are necessary for the correct folding and/or the stability of the NQR complex. Mass and EPR spectroscopy showed that NQR from V. harveyi bears only a 2Fe-2S cluster as a metal-containing prosthetic group. PMID:18298367

  8. Cluster conservation as a novel tool for studying protein-protein interactions evolution.

    PubMed

    Rahat, Ofer; Yitzhaky, Assif; Schreiber, Gideon

    2008-05-01

    Protein-protein interactions networks has come to be a buzzword associated with nets containing edges that represent a pair of interacting proteins (e.g. hormone-receptor, enzyme-inhibitor, antigen-antibody, and a subset of multichain biological machines). Yet, each such interaction composes its own unique network, in which vertices represent amino acid residues, and edges represent atomic contacts. Recent studies have shown that analyses of the data encapsulated in these detailed networks may impact predictions of structure-function correlation. Here, we study homologous families of protein-protein interfaces, which share the same fold but vary in sequence. In this context, we address what properties of the network are shared among relatives with different sequences (and hence different atomic interactions) and which are not. Herein, we develop the general mathematical framework needed to compare the modularity of homologous networks. We then apply this analysis to the structural data of a few interface families, including hemoglobin alpha-beta, growth hormone-receptor, and Serine protease-inhibitor. Our results suggest that interface modularity is an evolutionarily conserved property. Hence, protein-protein interfaces can be clustered down to a few modules, with the boundaries being evolutionarily conserved along homologous complexes. This suggests that protein engineering of protein-protein binding sites may be simplified by varying each module, but retaining the overall modularity of the interface. PMID:17972288

  9. MANAGING NITROGEN FOR COTTON IN A HIGH-RESIDUE CONSERVATION TILLAGE SYSTEM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Over 70% of the cotton (Gossypium hirsutum L.) in the Tennessee Valley of northern Alabama is currently raised using conservation tillage techniques. High-residue small grain cover crops are becoming a common tool in these systems, but N immobilization may occur causing previous N recommendations t...

  10. Cotton nitrogen management in a high-residue conservation system: source, rate, method, and timing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Over 70% of the 94,226 ha of cotton (Gossypium hirsutum L.) in the Tennessee Valley of northern Alabama, USA, is produced using conservation tillage systems with cereal cover crops. Decreased N efficiency, as a result of N immobilization and/or ammonia volatilization in high-residue systems, require...

  11. NITROGEN MANAGEMENT FOR COTTON GROWN IN A HIGH-RESIDUE COVER CROP CONSERVATION TILLAGE SYSTEM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Over 70% of the cotton (Gossypium hirsutum L.) in the Tennessee Valley of northern Alabama is currently raised using conservation tillage techniques. High-residue small grain cover crops are becoming a common tool in these systems, but N immobilization may occur causing previous N recommendations t...

  12. Unexpectedly large number of conserved noncoding regions within the ancestral chordate Hox cluster.

    PubMed

    Pascual-Anaya, Juan; D'Aniello, Salvatore; Garcia-Fernàndez, Jordi

    2008-12-01

    The single amphioxus Hox cluster contains 15 genes and may well resemble the ancestral chordate Hox cluster. We have sequenced the Hox genomic complement of the European amphioxus Branchiostoma lanceolatum and compared it to the American species, Branchiostoma floridae, by phylogenetic footprinting to gain insights into the evolution of Hox gene regulation in chordates. We found that Hox intergenic regions are largely conserved between the two amphioxus species, especially in the case of genes located at the 3' of the cluster, a trend previously observed in vertebrates. We further compared the amphioxus Hox cluster with the human HoxA, HoxB, HoxC, and HoxD clusters, finding several conserved noncoding regions, both in intergenic and intronic regions. This suggests that the regulation of Hox genes is highly conserved across chordates, consistent with the similar Hox expression patterns in vertebrates and amphioxus. PMID:18791732

  13. Conserved aromatic residues as determinants in the folding and assembly of immunoglobulin variable domains.

    PubMed

    Campion, Stephen R

    2016-02-01

    Detailed analysis of amino acid distribution, focusing on the "framework" regions of both heavy- and light-chain variable immunoglobulin (Ig) domains, distinguished those conserved sequence elements shared by both heavy-chain (VH) and light-chain (VL) domains from those conserved determinants unique to either VH or VL domains alone. Mapping of conserved chemical functionality onto characterized PDB structures showed the analogous placement and utilization of shared determinants in VH and VL structures that are generally similar. Identical Arginine-Aspartic acid ion-pairs located symmetrically on the lateral surfaces of VH and VL domains, respectively, as well as paired glutamine residues that constitute a central contact site between VH and VL domains represent clearly shared molecular features. Three sites of shared aromaticity were found localized to symmetrical sites lining the inaccessible interface of the VH-VL duplex, suggesting an expanded role for strategically conserved aromatic residues from a postulated determinant of individual Ig domain folding to now implicate conserved aromatic sites in the subsequent multi-subunit assembly of native antibody superstructure. Differential domain-specific conservation, representing evolutionary diversification and molecular asymmetry between heavy- and light-chain variable domains was limited, but included amino acids from each functional class and must be evaluated with regard to their possible involvement in heterologous aspects of IgV protein structure-function. PMID:26742085

  14. Conservation of Hox gene clusters in the self-fertilizing fish Kryptolebias marmoratus (Cyprinodontiformes; Rivulidae).

    PubMed

    Kim, B-M; Lee, B-Y; Lee, J-H; Rhee, J-S; Lee, J-S

    2016-03-01

    In this study, whole Hox gene clusters in the self-fertilizing mangrove killifish Kryptolebias marmoratus (Cyprinodontiformes; Rivulidae), a unique hermaphroditic vertebrate in which both sex organs are functional at the same time, were identified from whole genome and transcriptome sequences. The aim was to increase the understanding of the evolutionary status of conservation of this Hox gene cluster across fish species. PMID:26822496

  15. Multiple Roles of a Conserved GAF Domain Tyrosine Residue in Cyanobacterial and Plant Phytochromes†

    PubMed Central

    Fischer, Amanda J.; Rockwell, Nathan C.; Jang, Abigail Y.; Ernst, Lauren A.; Waggoner, Alan S.; Duan, Yong; Lei, Hongxing; Lagarias, J. Clark

    2005-01-01

    The phytochrome family of red/far-red photoreceptors has been optimized to support photochemical isomerization of a bound bilin chromophore, a process that triggers a conformational change and modulates biochemical output from the surrounding protein scaffold. Recent studies have established that the efficiency of this photochemical process is profoundly altered by mutation of a conserved tyrosine residue (Tyr176) within the bilin-binding GAF domain of the cyanobacterial phytochrome Cph1 [Fischer, A. J., and Lagarias, J. C. (2004) Harnessing phytochrome’s glowing potential, Proc. Natl. Acad. Sci. U.S.A. 101, 17334–17339]. Here, we show that the equivalent mutation in plant phytochromes behaves similarly, indicating that the function of this tyrosine in the primary photochemical mechanism is conserved. Saturation mutagenesis of Tyr176 in Cph1 establishes that no other residue can support comparably efficient photoisomerization. The spectroscopic consequences of Tyr176 mutations also reveal that Tyr176 regulates the conversion of the porphyrin-like conformation of the bilin precursor to a more extended conformation. The porphyrin-binding ability of the Tyr176Arg mutant protein indicates that Tyr176 also regulates the ligand-binding specificity of apophytochrome. On the basis of the hydrogen-bonding ability of Tyr176 substitutions that support the nonphotochemical C15-Z,syn to C15-Z,anti interconversion, we propose that Tyr176 orients the carboxyl side chain of a conserved acidic residue to stabilize protonation of the bilin chromophore. A homology model of the GAF domain of Cph1 predicts a C5-Z,syn, C10-Z,syn, C15-Z,anti configuration for the chromophore and implicates Glu189 as the proposed acidic residue stabilizing the extended conformation, an interpretation consistent with site-directed mutagenesis of this conserved acidic residue. PMID:16285723

  16. Conserved Residues of the Human Mitochondrial Holocytochrome c Synthase Mediate Interactions with Heme

    PubMed Central

    2015-01-01

    C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS–heme–cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical “correction” supports the proposed role of heme binding for the corresponding residues. PMID:25054239

  17. Contributions of conserved serine residues to the interactions of ligands with dopamine D2 receptors.

    PubMed

    Cox, B A; Henningsen, R A; Spanoyannis, A; Neve, R L; Neve, K A

    1992-08-01

    Four dopamine D2 receptor mutants were constructed, in each of which an alanine residue was substituted for one of four conserved serine residues, i.e., Ser-193, Ser-194, Ser-197, and Ser-391. Wild-type and mutant receptors were expressed transiently in COS-7 cells and stably in C6 glioma cells for analysis of ligand-receptor interactions. In radioligand binding assays, the affinity of D2 receptors for dopamine was decreased 50-fold by substitution of alanine for Ser-193, implicating this residue in the binding of dopamine. Each mutant had smaller decreases in affinity for one or more of the ligands tested, with no apparent relationship between the class of ligand and the pattern of mutation-induced changes in affinity, except that the potency of agonists was decreased by substitution for Ser-193. The potency of dopamine for inhibition of adenylyl cyclase was reduced substantially by substitution of alanine for Ser-193 or Ser-197. Mutation of Ser-194 led to a complete loss of efficacy for dopamine and p-tyramine, which would be consistent with an interaction between Ser-194 and the p-hydroxyl substituent of dopamine that is necessary for activation of the receptors to occur. Because mutation of the corresponding residues of beta 2-adrenergic receptors has very different consequences, we conclude that although the position of these serine residues is highly conserved among catecholamine receptors, and the residues as a group are important in ligand binding and activation of receptors by agonists, the function of each of the residues considered separately varies among catecholamine receptors. PMID:1321233

  18. Specific binding of eukaryotic ORC to DNA replication origins depends on highly conserved basic residues.

    PubMed

    Kawakami, Hironori; Ohashi, Eiji; Kanamoto, Shota; Tsurimoto, Toshiki; Katayama, Tsutomu

    2015-01-01

    In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC. PMID:26456755

  19. Specific binding of eukaryotic ORC to DNA replication origins depends on highly conserved basic residues

    PubMed Central

    Kawakami, Hironori; Ohashi, Eiji; Kanamoto, Shota; Tsurimoto, Toshiki; Katayama, Tsutomu

    2015-01-01

    In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC. PMID:26456755

  20. Search for conserved amino acid residues of the [Formula: see text]-crystallin proteins of vertebrates.

    PubMed

    Shiliaev, Nikita G; Selivanova, Olga M; Galzitskaya, Oxana V

    2016-04-01

    [Formula: see text]-crystallin is the major eye lens protein and a member of the small heat-shock protein (sHsp) family. [Formula: see text]-crystallins have been shown to support lens clarity by preventing the aggregation of lens proteins. We performed the bioinformatics analysis of [Formula: see text]-crystallin sequences from vertebrates to find conserved amino acid residues as the three-dimensional (3D) structure of [Formula: see text]-crystallin is not identified yet. We are the first who demonstrated that the N-terminal region is conservative along with the central domain for vertebrate organisms. We have found that there is correlation between the conserved and structured regions. Moreover, amyloidogenic regions also correspond to the structured regions. We analyzed the amino acid composition of [Formula: see text]-crystallin A and B chains. Analyzing the occurrence of each individual amino acid residue, we have found that such amino acid residues as leucine, serine, lysine, proline, phenylalanine, histidine, isoleucine, glutamic acid, and valine change their content simultaneously in A and B chains in different classes of vertebrates. Aromatic amino acids occur more often in [Formula: see text]-crystallins from vertebrates than on the average in proteins among 17 animal proteomes. We obtained that the identity between A and B chains in the mammalian group is 0.35, which is lower than the published 0.60. PMID:26972563

  1. Human immunodeficiency virus type 1 integrase: effect on viral replication of mutations at highly conserved residues.

    PubMed

    Cannon, P M; Wilson, W; Byles, E; Kingsman, S M; Kingsman, A J

    1994-08-01

    Sequence comparisons of the integrase (IN) proteins from different retroviruses have identified several highly conserved residues. We have introduced mutations at 16 of these sites into the integrase gene of human immunodeficiency virus type 1 and analyzed the phenotypes of the resulting viruses. The viruses were all normal for p24 content and reverse transcriptase activity. In addition, all of the mutants could infect T-cell lines and undergo reverse transcription, as assessed by PCR analysis. Most of the mutant viruses also had normal Western blot (immunoblot) profiles, although three of the mutations resulted in reduced signals for IN relative to the wild type on the immunoblots and mutation of residue W235 completely abolished recognition of the protein by pooled sera from human immunodeficiency virus type 1-positive patients. Mutations that have previously been shown to abolish activity in in vitro studies produced noninfectious viruses. The substitution of W235 was notable in producing a noninfectious virus, despite previous reports of this residue being nonessential for IN activity in vitro (A.D. Leavitt, L. Shiue, and H.E. Varmus, J. Biol. Chem. 268:2113-2119, 1993). In addition, we have identified four highly conserved residues that can be mutated without any affect on viral replication in T-cell lines. PMID:8035478

  2. WXG100 Protein Superfamily Consists of Three Subfamilies and Exhibits an α-Helical C-Terminal Conserved Residue Pattern

    PubMed Central

    Poulsen, Christian; Panjikar, Santosh; Holton, Simon J.; Wilmanns, Matthias; Song, Young-Hwa

    2014-01-01

    Members of the WXG100 protein superfamily form homo- or heterodimeric complexes. The most studied proteins among them are the secreted T-cell antigens CFP-10 (10 kDa culture filtrate protein, EsxB) and ESAT-6 (6 kDa early secreted antigen target, EsxA) from Mycobacterium tuberculosis. They are encoded on an operon within a gene cluster, named as ESX-1, that encodes for the Type VII secretion system (T7SS). WXG100 proteins are secreted in a full-length form and it is known that they adopt a four-helix bundle structure. In the current work we discuss the evolutionary relationship between the homo- and heterodimeric WXG100 proteins, the basis of the oligomeric state and the key structural features of the conserved sequence pattern of WXG100 proteins. We performed an iterative bioinformatics analysis of the WXG100 protein superfamily and correlated this with the atomic structures of the representative WXG100 proteins. We find, firstly, that the WXG100 protein superfamily consists of three subfamilies: CFP-10-, ESAT-6- and sagEsxA-like proteins (EsxA proteins similar to that of Streptococcus agalactiae). Secondly, that the heterodimeric complexes probably evolved from a homodimeric precursor. Thirdly, that the genes of hetero-dimeric WXG100 proteins are always encoded in bi-cistronic operons and finally, by combining the sequence alignments with the X-ray data we identify a conserved C-terminal sequence pattern. The side chains of these conserved residues decorate the same side of the C-terminal α-helix and therefore form a distinct surface. Our results lead to a putatively extended T7SS secretion signal which combines two reported T7SS recognition characteristics: Firstly that the T7SS secretion signal is localized at the C-terminus of T7SS substrates and secondly that the conserved residues YxxxD/E are essential for T7SS activity. Furthermore, we propose that the specific α-helical surface formed by the conserved sequence pattern including YxxxD/E motif is a key

  3. Conserved residues in the extracellular loops of short-wavelength cone visual pigments.

    PubMed

    Chen, Min-Hsuan; Sandberg, Daniel J; Babu, Kunnel R; Bubis, Jose; Surya, Arjun; Ramos, Lavoisier S; Zapata, Heidi J; Galan, Jhenny F; Sandberg, Megan N; Birge, Robert R; Knox, Barry E

    2011-08-16

    The role of the extracellular loop region of a short-wavelength sensitive pigment, Xenopus violet cone opsin, is investigated via computational modeling, mutagenesis, and spectroscopy. The computational models predict a complex H-bonding network that stabilizes and connects the EC2-EC3 loop and the N-terminus. Mutations that are predicted to disrupt the H-bonding network are shown to produce visual pigments that do not stably bind chromophore and exhibit properties of a misfolded protein. The potential role of a disulfide bond between two conserved Cys residues, Cys(105) in TM3 and Cys(182) in EC2, is necessary for proper folding and trafficking in VCOP. Lastly, certain residues in the EC2 loop are predicted to stabilize the formation of two antiparallel β-strands joined by a hairpin turn, which interact with the chromophore via H-bonding or van der Waals interactions. Mutations of conserved residues result in a decrease in the level of chromophore binding. These results demonstrate that the extracellular loops are crucial for the formation of this cone visual pigment. Moreover, there are significant differences in the structure and function of this region in VCOP compared to that in rhodopsin. PMID:21688771

  4. Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding.

    PubMed

    Barington, Line; Rummel, Pia C; Lückmann, Michael; Pihl, Heidi; Larsen, Olav; Daugvilaite, Viktorija; Johnsen, Anders H; Frimurer, Thomas M; Karlshøj, Stefanie; Rosenkilde, Mette M

    2016-07-29

    Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr(184) (Cys + 1) and Tyr(187) (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr(187) in TMIV (Phe(171)) and TMV (Trp(194)). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr(187) This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors. PMID:27226537

  5. A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

    PubMed Central

    2014-01-01

    Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of

  6. Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases

    PubMed Central

    Duncan, Anna L.

    2016-01-01

    The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme. PMID:27382158

  7. Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases.

    PubMed

    Duncan, Anna L; Robinson, Alan J; Walker, John E

    2016-08-01

    The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme. PMID:27382158

  8. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases.

    PubMed

    Salmon, Melissa; Thimmappa, Ramesha B; Minto, Robert E; Melton, Rachel E; Hughes, Richard K; O'Maille, Paul E; Hemmings, Andrew M; Osbourn, Anne

    2016-07-26

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  9. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

    PubMed Central

    Salmon, Melissa; Thimmappa, Ramesha B.; Minto, Robert E.; Melton, Rachel E.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne

    2016-01-01

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  10. Cob component of corn residue can be used as a biofuel feedstock with little impact on soil and water conservation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Use of corn residue as a biofuel feedstock raises a number of concerns related to soil and water conservation. Soil compaction, increased susceptibility for wind and water erosion, increased nutrient removal, and loss of soil organic matter are potential negative affects associated with residue remo...

  11. A cross-species bi-clustering approach to identifying conserved co-regulated genes

    PubMed Central

    Sun, Jiangwen; Jiang, Zongliang; Tian, Xiuchun; Bi, Jinbo

    2016-01-01

    Motivation: A growing number of studies have explored the process of pre-implantation embryonic development of multiple mammalian species. However, the conservation and variation among different species in their developmental programming are poorly defined due to the lack of effective computational methods for detecting co-regularized genes that are conserved across species. The most sophisticated method to date for identifying conserved co-regulated genes is a two-step approach. This approach first identifies gene clusters for each species by a cluster analysis of gene expression data, and subsequently computes the overlaps of clusters identified from different species to reveal common subgroups. This approach is ineffective to deal with the noise in the expression data introduced by the complicated procedures in quantifying gene expression. Furthermore, due to the sequential nature of the approach, the gene clusters identified in the first step may have little overlap among different species in the second step, thus difficult to detect conserved co-regulated genes. Results: We propose a cross-species bi-clustering approach which first denoises the gene expression data of each species into a data matrix. The rows of the data matrices of different species represent the same set of genes that are characterized by their expression patterns over the developmental stages of each species as columns. A novel bi-clustering method is then developed to cluster genes into subgroups by a joint sparse rank-one factorization of all the data matrices. This method decomposes a data matrix into a product of a column vector and a row vector where the column vector is a consistent indicator across the matrices (species) to identify the same gene cluster and the row vector specifies for each species the developmental stages that the clustered genes co-regulate. Efficient optimization algorithm has been developed with convergence analysis. This approach was first validated on

  12. A conserved aromatic residue regulating photosensitivity in short-wavelength sensitive cone visual pigments.

    PubMed

    Kuemmel, Colleen M; Sandberg, Megan N; Birge, Robert R; Knox, Barry E

    2013-07-30

    Visual pigments have a conserved phenylalanine in transmembrane helix 5 located near the β-ionone ring of the retinal chromophore. Site-directed mutants of this residue (F207) in a short-wavelength sensitive visual pigment (VCOP) were studied using UV-visible spectroscopy to investigate its role in photosensitivity and formation of the light-activated state. The side chain is important for pigment formation: VCOP(F207A), VCOP(F207L), VCOP(F207M), and VCOP(F207W) substitutions all bound 11-cis-retinal and formed a stable visual pigment, while VCOP(F207V), VCOP(F207S), VCOP(F207T), and VCOP(F207Y) substitutions do not. The extinction coefficients of all pigments are close, ranging between 35800 and 45600 M⁻¹ cm⁻¹. Remarkably, the mutants exhibit an up to 5-fold reduction in photosensitivity and also abnormal photobleaching behavior. One mutant, VCOP(F207A), forms an isomeric composition of the retinal chromophore after illumination comparable to that of wild-type VCOP yet does not release the all-trans-retinal chromophore. These findings suggest that the conserved F207 residue is important for a normal photoactivation pathway, formation of the active conformation and the exit of all-trans-retinal from the chromophore-binding pocket. PMID:23808485

  13. DID THE INFANT R136 AND NGC 3603 CLUSTERS UNDERGO RESIDUAL GAS EXPULSION?

    SciTech Connect

    Banerjee, Sambaran; Kroupa, Pavel E-mail: pavel@astro.uni-bonn.de

    2013-02-10

    Based on kinematic data observed for very young, massive clusters that appear to be in dynamical equilibrium, it has recently been argued that such young systems are examples of where the early residual gas expulsion did not happen or had no dynamical effect. The intriguing scenario of a star cluster forming through a single starburst has thereby been challenged. Choosing the case of the R136 cluster of the Large Magellanic Cloud, the most cited one in this context, we perform direct N-body computations that mimic the early evolution of this cluster including the gas-removal phase (on a thermal timescale). Our calculations show that under plausible initial conditions which are consistent with observational data, a large fraction (>60%) of a gas-expelled, expanding R136-like cluster is bound to regain dynamical equilibrium by its current age. Therefore, the recent measurements of velocity dispersion in the inner regions of R136, which indicate that the cluster is in dynamical equilibrium, are consistent with an earlier substantial gas expulsion of R136 followed by a rapid re-virialization (in Almost-Equal-To 1 Myr). Additionally, we find that the less massive Galactic NGC 3603 Young Cluster (NYC), with a substantially longer re-virialization time, is likely to be found to have deviated from dynamical equilibrium at its present age ( Almost-Equal-To 1 Myr). The recently obtained stellar proper motions in the central part of the NYC indeed suggest this and are consistent with the computed models. This work significantly extends previous models of the Orion Nebula Cluster which already demonstrated that the re-virialization time of young post-gas-expulsion clusters decreases with increasing pre-expulsion density.

  14. Did the Infant R136 and NGC 3603 Clusters Undergo Residual Gas Expulsion?

    NASA Astrophysics Data System (ADS)

    Banerjee, Sambaran; Kroupa, Pavel

    2013-02-01

    Based on kinematic data observed for very young, massive clusters that appear to be in dynamical equilibrium, it has recently been argued that such young systems are examples of where the early residual gas expulsion did not happen or had no dynamical effect. The intriguing scenario of a star cluster forming through a single starburst has thereby been challenged. Choosing the case of the R136 cluster of the Large Magellanic Cloud, the most cited one in this context, we perform direct N-body computations that mimic the early evolution of this cluster including the gas-removal phase (on a thermal timescale). Our calculations show that under plausible initial conditions which are consistent with observational data, a large fraction (>60%) of a gas-expelled, expanding R136-like cluster is bound to regain dynamical equilibrium by its current age. Therefore, the recent measurements of velocity dispersion in the inner regions of R136, which indicate that the cluster is in dynamical equilibrium, are consistent with an earlier substantial gas expulsion of R136 followed by a rapid re-virialization (in ≈1 Myr). Additionally, we find that the less massive Galactic NGC 3603 Young Cluster (NYC), with a substantially longer re-virialization time, is likely to be found to have deviated from dynamical equilibrium at its present age (≈1 Myr). The recently obtained stellar proper motions in the central part of the NYC indeed suggest this and are consistent with the computed models. This work significantly extends previous models of the Orion Nebula Cluster which already demonstrated that the re-virialization time of young post-gas-expulsion clusters decreases with increasing pre-expulsion density.

  15. Evolution of the transferrin family: conservation of residues associated with iron and anion binding.

    PubMed

    Lambert, Lisa A; Perri, Holly; Halbrooks, Peter J; Mason, Anne B

    2005-10-01

    The transferrin family spans both vertebrates and invertebrates. It includes serum transferrin, ovotransferrin, lactoferrin, melanotransferrin, inhibitor of carbonic anhydrase, saxiphilin, the major yolk protein in sea urchins, the crayfish protein, pacifastin, and a protein from green algae. Most (but not all) contain two domains of around 340 residues, thought to have evolved from an ancient duplication event. For serum transferrin, ovotransferrin and lactoferrin each of the duplicated lobes binds one atom of Fe (III) and one carbonate anion. With a few notable exceptions each iron atom is coordinated to four conserved amino acid residues: an aspartic acid, two tyrosines, and a histidine, while anion binding is associated with an arginine and a threonine in close proximity. These six residues in each lobe were examined for their evolutionary conservation in the homologous N- and C-lobes of 82 complete transferrin sequences from 61 different species. Of the ligands in the N-lobe, the histidine ligand shows the most variability in sequence. Also, of note, four of the twelve insect transferrins have glutamic acid substituted for aspartic acid in the N-lobe (as seen in the bacterial ferric binding proteins). In addition, there is a wide spread substitution of lysine for the anion binding arginine in the N-lobe in many organisms including all of the fish, the sea squirt and many of the unusual family members i.e., saxiphilin and the green alga protein. It is hoped that this short analysis will provide the impetus to establish the true function of some of the TF family members that clearly lack the ability to bind iron in one or both lobes and additionally clarify the evolutionary history of this important family of proteins. PMID:16111909

  16. Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein.

    PubMed

    Katsuma, Susumu; Sugano, Yudai; Kiuchi, Takashi; Shimada, Toru

    2015-10-23

    We have recently discovered that the Masculinizer (Masc) gene encodes a CCCH tandem zinc finger protein, which controls both masculinization and dosage compensation in the silkworm Bombyx mori. In this study, we attempted to identify functional regions or residues that are required for the masculinizing activity of the Masc protein. We constructed a series of plasmids that expressed the Masc derivatives and transfected them into a B. mori ovary-derived cell line, BmN-4. To assess the masculinizing activity of the Masc derivatives, we investigated the splicing patterns of B. mori doublesex (Bmdsx) and the expression levels of B. mori IGF-II mRNA-binding protein, a splicing regulator of Bmdsx, in Masc cDNA-transfected BmN-4 cells. We found that two zinc finger domains are not required for the masculinizing activity. We also identified that the C-terminal 288 amino acid residues are sufficient for the masculinizing activity of the Masc protein. Further detailed analyses revealed that two cysteine residues, Cys-301 and Cys-304, in the highly conserved region among lepidopteran Masc proteins are essential for the masculinizing activity in BmN-4 cells. Finally, we showed that Masc is a nuclear protein, but its nuclear localization is not tightly associated with the masculinizing activity. PMID:26342076

  17. Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

    PubMed

    Oliveira, Marco A S; Baura, Valter A; Aquino, Bruno; Huergo, Luciano F; Kadowaki, Marco A S; Chubatsu, Leda S; Souza, Emanuel M; Dixon, Ray; Pedrosa, Fábio O; Wassem, Roseli; Monteiro, Rose A

    2009-01-01

    Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA. PMID:19573596

  18. Identification and analysis of a highly conserved chemotaxis gene cluster in Shewanella species.

    SciTech Connect

    Li, J.; Romine, Margaret F.; Ward, M.

    2007-08-01

    A conserved cluster of chemotaxis genes was identified from the genome sequences of fifteen Shewanella species. An in-frame deletion of the cheA-3 gene, which is located in this cluster, was created in S. oneidensis MR-1 and the gene shown to be essential for chemotactic responses to anaerobic electron acceptors. The CheA-3 protein showed strong similarity to Vibrio cholerae CheA-2 and P. aeruginosa CheA-1, two proteins that are also essential for chemotaxis. The genes encoding these proteins were shown to be located in chemotaxis gene clusters closely related to the cheA-3-containing cluster in Shewanella species. The results of this study suggest that a combination of gene neighborhood and homology analyses may be used to predict which cheA genes are essential for chemotaxis in groups of closely related microorganisms.

  19. The roles of conserved and nonconserved cysteinyl residues in the oligomerization and function of mammalian prestin.

    PubMed

    Currall, Benjamin; Rossino, Danielle; Jensen-Smith, Heather; Hallworth, Richard

    2011-11-01

    The creation of several prestin knockout and knockin mouse lines has demonstrated the importance of the intrinsic outer hair cell membrane protein prestin to mammalian hearing. However, the structure of prestin remains largely unknown, with even its major features in dispute. Several studies have suggested that prestin forms homo-oligomers that may be stabilized by disulfide bonds. Our phylogenetic analysis of prestin sequences across chordate classes suggested that the cysteinyl residues could be divided into three groups, depending on the extent of their conservation between prestin orthologs and paralogs or homologs. An alanine scan functional analysis was performed of all nine cysteinyl positions in mammalian prestin. Prestin function was assayed by measurement of prestin-associated nonlinear capacitance. Of the nine cysteine-alanine substitution mutations, all were properly membrane targeted and all demonstrated nonlinear capacitance. Four mutations (C124A, C192A, C260A, and C415A), all in nonconserved cysteinyl residues, significantly differed in their nonlinear capacitance properties compared with wild-type prestin. In the two most severely disrupted mutations, substitution of the polar residue seryl for cysteinyl restored normal function in one (C415S) but not the other (C124S). We assessed the relationship of prestin oligomerization to cysteine position using fluorescence resonance energy transfer. With one exception, cysteine-alanine substitutions did not significantly alter prestin-prestin interactions. The exception was C415A, one of the two nonconserved cysteinyl residues whose mutation to alanine caused the most disruption in function. We suggest that no disulfide bond is essential for prestin function. However, C415 likely participates by hydrogen bonding in both nonlinear capacitance and oligomerization. PMID:21813750

  20. Characterization of Protective Epitopes in a Highly Conserved Plasmodium falciparum Antigenic Protein Containing Repeats of Acidic and Basic Residues

    PubMed Central

    Sharma, Pawan; Kumar, Anil; Singh, Balwan; Bharadwaj, Ashima; Sailaja, V. Naga; Adak, T.; Kushwaha, Ashima; Malhotra, Pawan; Chauhan, V. S.

    1998-01-01

    The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ∼90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum. PMID:9596765

  1. An insight to the conserved water mediated dynamics of catalytic His88 and its recognition to thyroxin and RBP binding residues in human transthyretin.

    PubMed

    Banerjee, Avik; Mukhopadhyay, Bishnu P

    2015-09-01

    Human transthyretin (hTTR) is a multifunctional protein involved in several amyloidogenic diseases. Besides transportation of thyroxin and vitamin-A, its role towards the catalysis of apolipoprotein-A1 and Aβ-peptide are also drawing interest. The role of water molecules in the catalytic mechanism is still unknown. Extensive analyses of 14 high-resolution X-ray structures of human transthyretin and MD simulation studies have revealed the presence of eight conserved hydrophilic centres near its catalytic zone which may be indispensable for the function, dynamics and stability of the protein. Three water molecules (W1, W2 and W3) form a cluster and play an important role in the recognition of the catalytic and RBP-binding residues. They also induce the reorganisation of the His88 for coupling with other catalytic residues (His90, Glu92). Another water molecule (W5) participate in inter-monomer recognition between the catalytic and thyroxin binding sites. The rest four water molecules (W6, W*, W(#) and W(†)) form a distorted tetrahedral cluster and impart stability to the catalytic core of hTTR. The conserved water mediated recognition dynamics of the different functional sites may provide some rational clues towards the understanding of the activity and mechanism of hTTR. PMID:25372987

  2. Understanding the roles of strictly conserved tryptophan residues in O2 producing chlorite dismutases

    PubMed Central

    Blanc, Beatrice; Rodgers, Kenton R.

    2013-01-01

    The chlorite dismutases (Clds) degrade ClO2− to O2 and Cl− in perchlorate respiring bacteria, and they serve still poorly defined cellular roles in other diverse microbes. These proteins share 3 highly conserved Trp residues, W155, W156, and W227, on the proximal side of the heme. The Cld from Dechloromonas aromatica (DaCld) has been shown to form protein-based radicals in its reactions with ClO2− and peracetic acid. The roles of the conserved Trp residues in radical generation and in enzymatic function were assessed via spectroscopic and kinetic analysis of their Phe mutants. The W155F mutant was the most dramatically affected, appearing to lose the characteristic pentameric oligomerization state, secondary structure, and heme binding properties of the WT protein. The W156F mutant initially retains many features of the WT protein but over time acquires many of the features of W155F. Conversion to an inactive, heme-free form is accelerated by dilution, suggesting loss of the protein’s pentameric state. Hence, both W155 and W156 are important for heme binding and maintenance of the protein’s reactive pentameric structure. W227F by contrast retains many properties of the WT protein. Important differences are noted in the transient kinetic reactions with peracetic acid (PAA), where W227F appears to form an [Fe(IV)=O]-containing intermediate, which subsequently converts to an uncoupled [Fe(IV)=O + AA+.] system in a [PAA]-dependent manner. This is in contrast to the peroxidase-like formation of [Fe(IV)=O] coupled to a porphyrin π-cation radical in the WT protein, which decays in a [PAA]-independent manner. These observations and the lack of redox protection for the heme in any of the Trp mutants suggests a tendency for protein radical formation in DaCld that is independent of any of these conserved active site residues. PMID:23241559

  3. The conserved protein Dre2 uses essential [2Fe-2S] and [4Fe-4S] clusters for its function in cytosolic iron-sulfur protein assembly.

    PubMed

    Netz, Daili J A; Genau, Heide M; Weiler, Benjamin D; Bill, Eckhard; Pierik, Antonio J; Lill, Roland

    2016-07-15

    The cytosolic iron-sulfur (Fe-S) protein assembly (CIA) machinery comprises 11 essential components and matures Fe-S proteins involved in translation and genome maintenance. Maturation is initiated by the electron transfer chain NADPH-diflavin reductase Tah18-Fe-S protein Dre2 that facilitates the de novo assembly of a [4Fe-4S] cluster on the scaffold complex Cfd1-Nbp35. Tah18-Dre2 also play a critical role in the assembly of the diferric tyrosyl radical cofactor of ribonucleotide reductase. Dre2 contains eight conserved cysteine residues as potential co-ordinating ligands for Fe-S clusters but their functional importance and the type of bound clusters is unclear. In the present study, we use a combination of mutagenesis, cell biological and biochemical as well as UV-visible, EPR and Mössbauer spectroscopic approaches to show that the yeast Dre2 cysteine residues Cys(252), Cys(263), Cys(266) and Cys(268) (motif I) bind a [2Fe-2S] cluster, whereas cysteine residues Cys(311), Cys(314), Cys(322) and Cys(325) (motif II) co-ordinate a [4Fe-4S] cluster. All of these residues with the exception of Cys(252) are essential for cell viability, cytosolic Fe-S protein activity and in vivo (55)Fe-S cluster incorporation. The N-terminal methyltransferase-like domain of Dre2 is important for proper Fe-S cluster assembly at motifs I and II, which occurs in an interdependent fashion. Our findings further resolve why recombinant Dre2 from Arabidopsis, Trypanosoma or humans has previously been isolated with a single [2Fe-2S] instead of native [2Fe-2S] plus [4Fe-4S] clusters. In the presence of oxygen, the motif I-bound [2Fe-2S] cluster is labile and the motif II-bound [4Fe-4S] cluster is readily converted into a [2Fe-2S] cluster. PMID:27166425

  4. Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain.

    PubMed

    Chen, Jingxin; Ray, Evan C; Yates, Megan E; Buck, Teresa M; Brodsky, Jeffrey L; Kinlough, Carol L; Winarski, Katie L; Hughey, Rebecca P; Kleyman, Thomas R; Sheng, Shaohu

    2015-10-01

    The extracellular regions of epithelial Na(+) channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na(+) (Na(+) self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na(+) channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na(+) self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na(+) self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na(+) self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na(+) self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs. PMID:26306034

  5. Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

    NASA Astrophysics Data System (ADS)

    da, Lin-Tai; Pardo-Avila, Fátima; Xu, Liang; Silva, Daniel-Adriano; Zhang, Lu; Gao, Xin; Wang, Dong; Huang, Xuhui

    2016-04-01

    The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3'-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3'-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.

  6. Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

    PubMed Central

    Da, Lin-Tai; Pardo-Avila, Fátima; Xu, Liang; Silva, Daniel-Adriano; Zhang, Lu; Gao, Xin; Wang, Dong; Huang, Xuhui

    2016-01-01

    The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation. PMID:27091704

  7. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    PubMed

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  8. Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthetase.

    PubMed

    Ghosh, G; Brunie, S; Schulman, L H

    1991-09-15

    The crystal structure of a fully biologically active monomeric form of Escherichia coli methionyl-tRNA synthetase (MetRS) complexed with ATP has recently been reported (Brunie, S., Zelwer, C., and Risler, J.-L., (1990) J. Mol. Biol. 216, 411-424), revealing details of the active site of the enzyme, including the location of amino acid residues potentially involved in substrate binding. In the present paper, the role of 3 active site residues in interaction with methionine, ATP, and tRNA(fMet) and in catalysis of methionyl-adenylate has been explored using site-directed mutagenesis. Lys142 is located near the ribose of ATP in the MetRS.ATP cocrystal. Mutation of this residue to Ala caused a 5-fold decrease in kcat/Km for ATP-PPi exchange, indicating some contribution of the lysine side chain to the specificity of the enzyme. Mutation of Tyr359 to Ala produced a 14-fold increase in the Km for ATP with only a small (2-3-fold) change in the other kinetic parameters, indicating that the major role of this residue is in formation of the initial complex with ATP and/or in stabilization of the methionyl-adenylate reaction intermediate. Mutation of the adjacent residue Tyr358 to Ala had no effect on the Km values for methionine or ATP but produced nearly a 2000-fold decrease in the rate of ATP-PPi exchange. This mutation also dramatically reduced the rate of pyrophosphorolysis of the isolated MetRS.Met-AMP complex on addition of pyrophosphate without increasing the Km for PPi. None of the mutations affected the Km for tRNAfMet in the aminoacylation reaction. The results suggest that Tyr358 may enhance the rate of methionyl-adenylate formation by binding to the alpha-phosphate of ATP in the transition state. Interaction of Tyr358 and Tyr359 with ATP during the course of the reaction requires a significant change in the conformation of this region of the active site compared to the structure found in the MetRS.ATP complex. Such a shift is consistent with an induced

  9. Site-directed mutagenesis of a conserved Asn450 residue of Bacillus licheniformis γ-glutamyltranspeptidase.

    PubMed

    Lin, Min-Guan; Chi, Meng-Chun; Chen, Yu-Yi; Wang, Tzu-Fan; Lo, Hui-Fen; Lin, Long-Liu

    2016-10-01

    Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) belongs to N-terminal nucleophile hydrolase superfamily in which all inclusive members are synthetized as single-chain precursors, and then self-processed to form mature enzymes. Here we investigated the role of a conserved Asn450 residue in BlGGT through site-directed mutagenesis and molecular characterization of four relevant variants. Substitution of Asn450 by arginine resulted in a significant reduction in the catalytic activity of BlGGT. Conversely, N450A and N450D displayed an enhanced activity. The catalytic efficiency of BlGGT was calculated to be 16.04mM(-1)s(-1), but this value was either decreased to 8.93mM(-1)s(-1) in N450K or increased to more than 123.65mM(-1)s(-1) in N450A and N450D. In addition, the ratio of transpeptidation to hydrolysis was increased from 3.5 to more than 7.6 by the mutations. Structural analyses showed that fluorescence, circular dichroism spectra and thermal denaturation profiles of mutant proteins were essentially consistent with those of BlGGT. However, guanidine hydrochloride (GdnHCl)-induced transition was significantly reduced in comparison with the wild-type enzyme. Molecular modeling suggests that residue Asn450 of BlGGT is important to create suitable environments for both autoprocessing and catalytic reactions. PMID:27246377

  10. Manipulating Conserved Heme Cavity Residues of Chlorite Dismutase: Effect on Structure, Redox Chemistry, and Reactivity

    PubMed Central

    2013-01-01

    Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from “Candidatus Nitrospira defluvii” (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV–vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O=O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E°′ = −113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation. PMID:24364531

  11. Interfacial partitioning of a loop hinge residue contributes to diacylglycerol affinity of conserved region 1 domains.

    PubMed

    Stewart, Mikaela D; Cole, Taylor R; Igumenova, Tatyana I

    2014-10-01

    Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant. The W252Y mutation was previously shown to render C1Bδ less responsive to DAG (Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J. Biol. Chem. 282, 826-830) and thereby emulate the behavior of C1B domains from conventional PKCs that have a conserved Tyr at the equivalent position. Our data revealed that W252Y mutation did not perturb the conformation of C1Bδ in solution but significantly reduced its propensity to partition into a membrane-mimicking environment in the absence of DAG. Using detergent micelles doped with a paramagnetic lipid, we determined that both the residue identity at position 252 and complexation with diacylglycerol influence the geometry of C1Bδ-micelle interactions. In addition, we identified the C-terminal helix α1 of C1Bδ as an interaction site with the head groups of phosphatidylserine, a known activator of PKCδ. Taken together, our studies (i) reveal the identities of C1Bδ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities. PMID:25124034

  12. Interfacial Partitioning of a Loop Hinge Residue Contributes to Diacylglycerol Affinity of Conserved Region 1 Domains*

    PubMed Central

    Stewart, Mikaela D.; Cole, Taylor R.; Igumenova, Tatyana I.

    2014-01-01

    Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant. The W252Y mutation was previously shown to render C1Bδ less responsive to DAG (Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J. Biol. Chem. 282, 826–830) and thereby emulate the behavior of C1B domains from conventional PKCs that have a conserved Tyr at the equivalent position. Our data revealed that W252Y mutation did not perturb the conformation of C1Bδ in solution but significantly reduced its propensity to partition into a membrane-mimicking environment in the absence of DAG. Using detergent micelles doped with a paramagnetic lipid, we determined that both the residue identity at position 252 and complexation with diacylglycerol influence the geometry of C1Bδ-micelle interactions. In addition, we identified the C-terminal helix α1 of C1Bδ as an interaction site with the head groups of phosphatidylserine, a known activator of PKCδ. Taken together, our studies (i) reveal the identities of C1Bδ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities. PMID:25124034

  13. Conservation of a vitellogenin gene cluster in oviparous vertebrates and identification of its traces in the platypus genome.

    PubMed

    Babin, Patrick J

    2008-04-30

    Vitellogenin (Vtg) derivatives are the main egg-yolk proteins in most oviparous animal species, and are, therefore, key players in reproduction and embryo development. Conserved synteny and phylogeny were used to identify a Vtg gene cluster (VGC) that had been evolutionarily conserved in most oviparous vertebrates, encompassing the three linked Vtgs on chicken (Gallus gallus) chromosome 8. Tandem arranged homologs to chicken VtgII and VtgIII were retrieved in similar locations in Xenopus (Xenopus tropicalis) and homologous transcribed inverted genes were found in medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), pufferfish (Takifugu rubripes), and Tetrahodon (Tetraodon nigroviridis), while zebrafish (Danio rerio) Vtg3 may represent a residual trace of VGC in this genome. Vtgs were not conserved in the paralogous chromosomal segment attributed to a whole-genome duplication event in the ancestor of teleosts, while tandem duplicated forms have survived the recent African clawed frog (Xenopus laevis) tetraploidization. Orthologs to chicken VtgI were found in similar locations in teleost fish, as well as in the platypus (Ornithorhynchus anatinus). Additional Vtg fragments found suggested that VGC had been conserved in this egg-laying mammal. A low ratio of nonsynonymous-to-synonymous substitution values and the paucity of pseudogene features suggest functional platypus Vtg products. Genomic identification of Vtgs, Apob, and Mtp in this genome, together with maximum likelihood and Bayesian inference phylogenetic analyses, support the existence of these three large lipid transfer protein superfamily members at the base of the mammalian lineage. In conclusion, the establishment of a VGC in the vertebrate lineage predates the divergence of ray-finned fish and tetrapods and the shift in reproductive and developmental strategy observed between prototherians and therians may be associated with its loss, as shown by its absence from the genomic resources currently

  14. Mutagenesis of conserved active site residues of dihydrolipoamide succinyltransferase enhances the accumulation of α-ketoglutarate in Yarrowia lipolytica.

    PubMed

    Guo, Hongwei; Madzak, Catherine; Du, Guocheng; Zhou, Jingwen

    2016-01-01

    α-Ketoglutarate (α-KG) is an important intermediate in the tricarboxylic acid cycle and has broad applications. The mitochondrial ketoglutarate dehydrogenase (KGDH) complex catalyzes the oxidation of α-KG to succinyl-CoA. Disruption of KGDH, which may enhance the accumulation of α-KG theoretically, was found to be lethal to obligate aerobic cells. In this study, individual overexpression of dihydrolipoamide succinyltransferase (DLST), which serves as the inner core of KGDH, decreased overall activity of the enzyme complex. Furthermore, two conserved active site residues of DLST, His419, and Asp423 were identified. In order to determine whether these residues are engaged in enzyme reaction or not, these two conserved residues were individually mutated. Analysis of the kinetic parameters of the enzyme variants provided evidence that the catalytic reaction of DLST depended on residues His419 and Asp423. Overexpression of mutated DLST not only impaired balanced assembly of KGDH, but also disrupted the catalytic integrity of the enzyme complex. Replacement of the Asp423 residue by glutamate increased extracellular α-KG by 40 % to 50 g L(-1) in mutant strain. These observations uncovered catalytic roles of two conserved active site residues of DLST and provided clues for effective metabolic strategies for rational carbon flux control for the enhanced production of α-KG and related bioproducts. PMID:26428234

  15. MetaMirClust: Discovery and Exploration of Evolutionarily Conserved miRNA Clusters.

    PubMed

    Chan, Wen-Ching; Lin, Wen-Chang

    2016-01-01

    Recent emerging studies suggest that a substantial fraction of microRNA (miRNA) genes is likely to form clusters in terms of evolutionary conservation and biological implications, posing a significant challenge for the research community and shifting the bottleneck of scientific discovery from miRNA singletons to miRNA clusters. In addition, the advance in molecular sequencing technique such as next-generation sequencing (NGS) has facilitated researchers to comprehensively characterize miRNAs with low abundance on genome-wide scale in multiple species. Taken together, a large scale, cross-species survey of grouped miRNAs based on genomic location would be valuable for investigating their biological functions and regulations in an evolutionary perspective. In the present chapter, we describe the application of effective and efficient bioinformatics tools on the identification of clustered miRNAs and illustrate how to use the recently developed Web-based database, MetaMirClust ( http://fgfr.ibms.sinic.aedu.tw/MetaMirClust ) to discover evolutionarily conserved pattern of miRNA clusters across metazoans. PMID:25861770

  16. Evidence that Highly Conserved Residues of Transmembrane Segment 6 of Escherichia coli MntH Are Important for Transport Activity

    PubMed Central

    Haemig, Heather A.H.; Moen, Patrick J.; Brooker, Robert J.

    2010-01-01

    Nramp (natural resistance-associated macrophage protein) family members have been characterized in mammals, yeast, and bacteria as divalent metal ion/H+ symporters. In previous work, a bioinformatic approach was used for the identification of residues that are conserved within the Nramp family [ref.1. Haemig, H.A. and R.J. Brooker, (2004) J Membr. Biol, 201(2): 97-107]. Based on site-directed mutagenesis of highly conserved negatively charged residues, a model was proposed for the metal binding site of the E.coli homolog, MntH. In this study, we have focused on the highly conserved residues, including two histidines, of transmembrane segment 6 (TMS-6). Multiple mutants were made at the eight conserved sites (i.e., Gly-205, Ala-206, Met-209, Pro-210, His-211, Leu-215, His-216, and Ser-217) in TMS-6 of E. coli MntH. Double mutants involving His-211 and His-216 were also made. The results indicate the side chain volume of these residues is critically important for function. In most cases, only substitutions that are closest in side chain volume still permit transport. In addition, the Km for metal binding is largely unaffected by mutations in TMS-6, whereas Vmax values were decreased in all mutants characterized kinetically. Thus, these residues do not appear to play a role in metal binding. Instead, they may comprise an important face on TMS-6 that is critical for protein conformational changes during transport. Also, in contrast to other studies, our data do not strongly indicate that the conserved histidine residues play a role in pH regulation of metal transport. PMID:20441230

  17. Potential of high residue conservation tillage to enhance water conservation and water use efficiency in corn production in the Southeast

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Following the adoption of its first Comprehensive State-wide Water Management Plan in early February 2008, Georgia is on course to drafting regionally-based water development and conservation plans. Conservation tillage-based crop production can be one of the management tools available for achieving...

  18. Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase.

    PubMed

    Ranjani, Velayudhan; Janeček, Stefan; Chai, Kian Piaw; Shahir, Shafinaz; Abdul Rahman, Raja Noor Zaliha Raja; Chan, Kok-Gan; Goh, Kian Mau

    2014-01-01

    The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, k(cat) and k(cat)/K(m) higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA. PMID:25069018

  19. Conserved cysteine residues in the pore region are obligatory for human TRPM2 channel function

    PubMed Central

    Mei, Zhu-Zhong; Mao, Hong-Ju; Jiang, Lin-Hua

    2006-01-01

    TRPM2 proteins belong to the melastatin-related transient receptor potential or TRPM subfamily and form Ca2+-permeable cationic channels activated by intracellular adenosine diphosphoribose (ADPR). The TRPM2 channel subunit, like all its close relatives, is structurally homologous to the well-characterized voltage-gated potassium channel subunits, each containing six transmembrane segments and a putative pore loop between the fifth and sixth segments. Nevertheless, the structural elements determining the TRPM2 channel functions are still not well understood. In this study, we investigated the functional role of two conserved cysteine residues (at positions 996 and 1008) in the putative pore region of the human TRPM2 by site-directed mutagenesis combined with electrophysiological and biochemical approaches. Expression of wild type hTRPM2 channels in HEK293 cells resulted in robust ADPR-evoked currents. Substitution of cysteine with alanine or serine generated mutant channels that failed to be activated by ADPR. Furthermore, experiments by Western blotting, immunocytochemistry, biotin labelling, and co-immunoprecipitation techniques showed no obvious changes in protein expression, trafficking or membrane localisation, and the ability of interacting with neighbouring subunits that is required for channel assembly. Co-expression of wild type and mutant subunits significantly reduced the ADPR-evoked currents; for combination of wild type and C996S mutant subunits, the reduction was approximately 95%, indicating that incorporation of one or more non-functional C996S subunits leads to the loss of channel function. These results taken together suggest that the cysteine residues in the pore region are obligatory for TRPM2 channel function. PMID:16822940

  20. The role of a conserved tyrosine residue in high-potential iron sulfur proteins.

    PubMed Central

    Iwagami, S. G.; Creagh, A. L.; Haynes, C. A.; Borsari, M.; Felli, I. C.; Piccioli, M.; Eltis, L. D.

    1995-01-01

    Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring. PMID:8580847

  1. The conserved active-site loop residues of ferrochelatase induce porphyrin conformational changes necessary for catalysis.

    SciTech Connect

    Haddad, Raid Edward; Shelnutt, John Allen; Shi, Zhen; Ferreira, Gloria C.; Franco, Ricardo T.

    2005-05-01

    Binding of porphyrin to murine ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is investigated by employing a set of variants harboring mutations in a putative porphyrin-binding loop. Using resonance Raman (RR) spectroscopy, the structural properties of the ferrochelatase-bound porphyrins are examined, especially with respect to the porphyrin deformation occurring in the environment of the active site. This deformation is thought to be a key step in the enzymatic insertion of ferrous iron into the porphyrin ring to make heme. Our previous RR spectroscopic studies of binding of porphyrin to murine ferrochelatase led us to propose that the wild-type enzyme induces porphyrin distortion even in the absence of the metal ion substrate. Here, we broaden this view by presenting evidence that the degree of a specific nonplanar porphyrin deformation contributes to the catalytic efficiency of ferrochelatase and its variants. The results also suggest that the conserved Trp256 (murine ferrochelatase numbering) is partially responsible for the observed porphyrin deformation. Binding of porphyrin to the ferrochelatase variants causes a decrease in the intensity of RR out-of-plane vibrational mode {gamma}{sub 15}, a saddling-like mode that is strong in the wild-type enzyme. In particular, the variant with a catalytic efficiency 1 order of magnitude lower than that of the wild-type enzyme is estimated to produce less than 30% of the wild-type saddling deformation. These results suggest that specific conserved loop residues (especially Trp256) are directly involved in the saddling of the porphyrin substrate.

  2. Identification of an Ideal-like Fingerprint for a Protein Fold using Overlapped Conserved Residues based Approach

    PubMed Central

    Goyal, Amit; Sokalingam, Sriram; Hwang, Kyu-Suk; Lee, Sun-Gu

    2014-01-01

    Design of an efficient fingerprint that detects homologous proteins at distant sequence identity has been a great challenge. This paper proposes a strategy to extract an ideal-like fingerprint with high specificity and sensitivity from a group of sequences related to a fold. The approach is devised based on the assumptions that the critical residues for a protein fold may be conserved in three aspects, i.e. sequence, structure, and intramolecular interaction, and embedded in secondary structures. We hypothesized that the residues satisfying such conditions simultaneously may work as an efficient fingerprint. This idea was tested on protein folds of various classes, such as beta-strand rich, alpha + beta proteins and alpha/beta proteins with discrete sequence similarities. The fingerprint for each fold was generated by selecting the overlapped conserved residues (OCR) from the conserved residues obtained using independent three alignment methods, i.e. multiple sequence alignment, structure-based alignment, and alignment based on the interstrand hydrogen-bonds. The OCR fingerprints showed more than 90% detection efficiency for all the folds tested and were identified to be almost the minimal fingerprints composed of only critical residues. This study is expected to provide an important conceptual improvement in the identification or design of ideal fingerprints for a protein fold. PMID:25008052

  3. The role of conserved inulosucrase residues in the reaction and product specificity of Lactobacillus reuteri inulosucrase.

    PubMed

    Anwar, Munir A; Leemhuis, Hans; Pijning, Tjaard; Kralj, Slavko; Dijkstra, Bauke W; Dijkhuizen, Lubbert

    2012-10-01

    The probiotic bacterium Lactobacillus reuteri 121 produces two fructosyltransferase enzymes, a levansucrase and an inulosucrase. Although these two fructosyltransferase enzymes share high sequence similarity, they differ significantly in the type and size distribution of fructooligosaccharide products synthesized from sucrose, and in their activity levels. In order to examine the contribution of specific amino acids to such differences, 15 single and four multiple inulosucrase mutants were designed that affected residues that are conserved in inulosucrase enzymes, but not in levansucrase enzymes. The effects of the mutations were interpreted using the 3D structures of Bacillus subtilis levansucrase (SacB) and Lactobacillus johnsonii inulosucrase (InuJ). The wild-type inulosucrase synthesizes mostly fructooligosaccharides up to a degree of polymerization of 15 and relatively low amounts of inulin polymer. In contrast, wild-type levansucrase produces mainly levan polymer and fructooligosaccharides with a degree of polymerization < 5. Although most of the inulosucrase mutants in this study behaved similarly to the wild-type enzyme, the mutation G416E, at the rim of the active site pocket in loop 415-423, increased the hydrolytic activity twofold, without significantly changing the transglycosylation activity. The septuple mutant GM4 (T413K, K415R, G416E, A425P, S442N, W486L, P516L), which included two residues from the above-mentioned loop 415-423, synthesized 1-kestose only, but at low efficiency. Mutation A538S, located behind the general acid/base, increased the enzyme activity two to threefold. Mutation N543S, located adjacent to the +1/+2 sub-site residue R544, resulted in synthesis of not such a wide variety of fructooligosaccharides than the wild-type enzyme. The present study demonstrates that the product specificity of inulosucrase is easily altered by protein engineering, obtaining inulosucrase variants with higher transglycosylation specificity, higher

  4. Production of bioactive diterpenoids in the euphorbiaceae depends on evolutionarily conserved gene clusters.

    PubMed

    King, Andrew J; Brown, Geoffrey D; Gilday, Alison D; Larson, Tony R; Graham, Ian A

    2014-08-01

    The Euphorbiaceae produce a diverse range of diterpenoids, many of which have pharmacological activities. These diterpenoids include ingenol mebutate, which is licensed for the treatment of a precancerous skin condition (actinic keratosis), and phorbol derivatives such as resiniferatoxin and prostratin, which are undergoing investigation for the treatment of severe pain and HIV, respectively. Despite the interest in these diterpenoids, their biosynthesis is poorly understood at present, with the only characterized step being the conversion of geranylgeranyl pyrophosphate into casbene. Here, we report a physical cluster of diterpenoid biosynthetic genes from castor (Ricinus communis), including casbene synthases and cytochrome P450s from the CYP726A subfamily. CYP726A14, CYP726A17, and CYP726A18 were able to catalyze 5-oxidation of casbene, a conserved oxidation step in the biosynthesis of this family of medicinally important diterpenoids. CYP726A16 catalyzed 7,8-epoxidation of 5-keto-casbene and CYP726A15 catalyzed 5-oxidation of neocembrene. Evidence of similar gene clustering was also found in two other Euphorbiaceae, including Euphorbia peplus, the source organism of ingenol mebutate. These results demonstrate conservation of gene clusters at the higher taxonomic level of the plant family and that this phenomenon could prove useful in further elucidating diterpenoid biosynthetic pathways. PMID:25172144

  5. Sequence-related human proteins cluster by degree of evolutionary conservation

    NASA Astrophysics Data System (ADS)

    Mrowka, Ralf; Patzak, Andreas; Herzel, Hanspeter; Holste, Dirk

    2004-11-01

    Gene duplication followed by adaptive evolution is thought to be a central mechanism for the emergence of novel genes. To illuminate the contribution of duplicated protein-coding sequences to the complexity of the human genome, we study the connectivity of pairwise sequence-related human proteins and construct a network (N) of linked protein sequences with shared similarities. We find that (i) the connectivity distribution P(k) for k sequence-related proteins decays as a power law P(k)˜k-γ with γ≈1.2 , (ii) the top rank of N consists of a single large cluster of proteins (≈70%) , while bottom ranks consist of multiple isolated clusters, and (iii) structural characteristics of N show both a high degree of clustering and an intermediate connectivity (“small-world” features). We gain further insight into structural properties of N by studying the relationship between the connectivity distribution and the phylogenetic conservation of proteins in bacteria, plants, invertebrates, and vertebrates. We find that (iv) the proportion of sequence-related proteins increases with increasing extent of evolutionary conservation. Our results support that small-world network properties constitute a footprint of an evolutionary mechanism and extend the traditional interpretation of protein families.

  6. Conserved residues in the HAMP domain define a new family of proposed bipartite energy taxis receptors.

    PubMed

    Elliott, Kathryn T; Zhulin, Igor B; Stuckey, Jeanne A; DiRita, Victor J

    2009-01-01

    HAMP domains, found in many bacterial signal transduction proteins, generally transmit an intramolecular signal between an extracellular sensory domain and an intracellular signaling domain. Studies of HAMP domains in proteins where both the input and output signals occur intracellularly are limited to those of the Aer energy taxis receptor of Escherichia coli, which has both a HAMP domain and a sensory PAS domain. Campylobacter jejuni has an energy taxis system consisting of the domains of Aer divided between two proteins, CetA (HAMP domain containing) and CetB (PAS domain containing). In this study, we found that the CetA HAMP domain differs significantly from that of Aer in the predicted secondary structure. Using similarity searches, we identified 55 pairs of HAMP/PAS proteins encoded by adjacent genes in a diverse group of microorganisms. We propose that these HAMP/PAS pairs form a new family of bipartite energy taxis receptors. Within these proteins, we identified nine residues in the HAMP domain and proximal signaling domain that are highly conserved, at least three of which are required for CetA function. Additionally, we demonstrated that CetA contributes to the invasion of human epithelial cells by C. jejuni, while CetB does not. This finding supports the hypothesis that members of HAMP/PAS pairs possess the capacity to act independently of each other in cellular traits other than energy taxis. PMID:18952801

  7. Role of Conserved Proline Residues in Human Apolipoprotein A-IV Structure and Function*

    PubMed Central

    Deng, Xiaodi; Walker, Ryan G.; Morris, Jamie; Davidson, W. Sean; Thompson, Thomas B.

    2015-01-01

    Apolipoprotein (apo)A-IV is a lipid emulsifying protein linked to a range of protective roles in obesity, diabetes, and cardiovascular disease. It exists in several states in plasma including lipid-bound in HDL and chylomicrons and as monomeric and dimeric lipid-free/poor forms. Our recent x-ray crystal structure of the central domain of apoA-IV shows that it adopts an elongated helical structure that dimerizes via two long reciprocating helices. A striking feature is the alignment of conserved proline residues across the dimer interface. We speculated that this plays important roles in the structure of the lipid-free protein and its ability to bind lipid. Here we show that the systematic conversion of these prolines to alanine increased the thermodynamic stability of apoA-IV and its propensity to oligomerize. Despite the structural stabilization, we noted an increase in the ability to bind and reorganize lipids and to promote cholesterol efflux from cells. The novel properties of these mutants allowed us to isolate the first trimeric form of an exchangeable apolipoprotein and characterize it by small-angle x-ray scattering and chemical cross-linking. The results suggest that the reciprocating helix interaction is a common feature of all apoA-IV oligomers. We propose a model of how self-association of apoA-IV can result in spherical lipoprotein particles, a model that may have broader applications to other exchangeable apolipoprotein family members. PMID:25733664

  8. Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase.

    PubMed

    Robbins, Thomas; Kapilivsky, Joshuah; Cane, David E; Khosla, Chaitan

    2016-08-16

    Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C-C bond formation. S315 played a role in coupling decarboxylation to C-C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs. PMID:27441852

  9. The Respiratory Arsenite Oxidase: Structure and the Role of Residues Surrounding the Rieske Cluster

    PubMed Central

    Warelow, Thomas P.; Oke, Muse; Schoepp-Cothenet, Barbara; Dahl, Jan U.; Bruselat, Nicole; Sivalingam, Ganesh N.; Leimkühler, Silke; Thalassinos, Konstantinos; Kappler, Ulrike; Naismith, James H.; Santini, Joanne M.

    2013-01-01

    The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large α subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small β subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a −20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc1 complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc1 complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter. PMID:24023621

  10. The respiratory arsenite oxidase: structure and the role of residues surrounding the rieske cluster.

    PubMed

    Warelow, Thomas P; Oke, Muse; Schoepp-Cothenet, Barbara; Dahl, Jan U; Bruselat, Nicole; Sivalingam, Ganesh N; Leimkühler, Silke; Thalassinos, Konstantinos; Kappler, Ulrike; Naismith, James H; Santini, Joanne M

    2013-01-01

    The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large α subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small β subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a -20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc 1 complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc 1 complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter. PMID:24023621

  11. Conserved Active Site Residues Limit Inhibition of a Copper-Containing Nitrite By Small Molecules

    SciTech Connect

    Tocheva, E.I.; Eltis, L.D.; Murphy, M.E.P.

    2009-05-26

    The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.

  12. The Conserved Arginine Cluster in the Insert of the Third Cytoplasmic Loop of the Long Form of the D₂ Dopamine Receptor (D2L-R) Acts as an Intracellular Retention Signal.

    PubMed

    Kubale, Valentina; Blagotinšek, Kaja; Nøhr, Jane; Eidne, Karin A; Vrecl, Milka

    2016-01-01

    This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D₂ dopamine receptor (D2L-R) confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR) located within the insert could act as an RXR-type endoplasmic reticulum (ER) retention signal. Arginine residues (R) within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E), either individually or in clusters, thus generating single, double, and triple D2L-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA), radioligand binding assay, bioluminescence resonance energy transfer (BRET²) β-arrestin 2 (βarr2) recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E) substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D2L-R mutants preserved their functional integrity regarding ligand binding, agonist-induced βarr2 recruitment and Gαi-mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3) of the D2L-R appears to be the ER retention signal. PMID:27447620

  13. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  14. Conserved gene clusters in bacterial genomes provide further support for the primacy of RNA

    NASA Technical Reports Server (NTRS)

    Siefert, J. L.; Martin, K. A.; Abdi, F.; Widger, W. R.; Fox, G. E.

    1997-01-01

    Five complete bacterial genome sequences have been released to the scientific community. These include four (eu)Bacteria, Haemophilus influenzae, Mycoplasma genitalium, M. pneumoniae, and Synechocystis PCC 6803, as well as one Archaeon, Methanococcus jannaschii. Features of organization shared by these genomes are likely to have arisen very early in the history of the bacteria and thus can be expected to provide further insight into the nature of early ancestors. Results of a genome comparison of these five organisms confirm earlier observations that gene order is remarkably unpreserved. There are, nevertheless, at least 16 clusters of two or more genes whose order remains the same among the four (eu)Bacteria and these are presumed to reflect conserved elements of coordinated gene expression that require gene proximity. Eight of these gene orders are essentially conserved in the Archaea as well. Many of these clusters are known to be regulated by RNA-level mechanisms in Escherichia coli, which supports the earlier suggestion that this type of regulation of gene expression may have arisen very early. We conclude that although the last common ancestor may have had a DNA genome, it likely was preceded by progenotes with an RNA genome.

  15. Discovery of five conserved β-defensin gene clusters using a computational search strategy

    PubMed Central

    Schutte, Brian C.; Mitros, Joseph P.; Bartlett, Jennifer A.; Walters, Jesse D.; Jia, Hong Peng; Welsh, Michael J.; Casavant, Thomas L.; McCray, Paul B.

    2002-01-01

    The innate immune system includes antimicrobial peptides that protect multicellular organisms from a diverse spectrum of microorganisms. β-Defensins comprise one important family of mammalian antimicrobial peptides. The annotation of the human genome fails to reveal the expected diversity, and a recent query of the draft sequence with the blast search engine found only one new β-defensin gene (DEFB3). To define better the β-defensin gene family, we adopted a genomics approach that uses hmmer, a computational search tool based on hidden Markov models, in combination with blast. This strategy identified 28 new human and 43 new mouse β-defensin genes in five syntenic chromosomal regions. Within each syntenic cluster, the gene sequences and organization were similar, suggesting each cluster pair arose from a common ancestor and was retained because of conserved functions. Preliminary analysis indicates that at least 26 of the predicted genes are transcribed. These results demonstrate the value of a genomewide search strategy to identify genes with conserved structural motifs. Discovery of these genes represents a new starting point for exploring the role of β-defensins in innate immunity. PMID:11854508

  16. Evolutionary conservation and function of the human embryonic stem cell specific miR-302/367 cluster.

    PubMed

    Chen, Liang; Heikkinen, Liisa; Emily Knott, K; Liang, Yanchun; Wong, Garry

    2015-12-01

    miRNA clusters define a group of related miRNAs closely localized in the genome with an evolution that remains poorly understood. The miR-302/367 cluster represents a single polycistronic transcript that produces five precursor miRNAs. The cluster is highly expressed and essential for maintenance of human embryonic stem cells. We found the cluster to be highly conserved and present in most mammals. In primates, seed sequence and miRNA structure are conserved, but inter-precursor sequences are evolving. Insertions of new miRNAs, deletions of individual miRNAs, and a cluster duplication observed in different species suggest an actively evolving cluster. Core transcriptional machinery consisting of NANOG and OCT-4 transcription factors that define stem cells are present upstream of the miR-302/367 cluster. Interestingly, we found the miR-302/367 cluster flanking region to be enriched as a target site of other miRNAs suggesting a mechanism for feedback control. Analysis of miR-302 and miR-367 targets demonstrated concordance of gene set enrichment groups at high gene ontology levels. This cluster also expresses isomiRs providing another means of establishing sequence diversity. Finally, using three different kidney tumor datasets, we observed consistent expression of miR-302 family members in normal tissue while adjacent tumor tissue showed a significant lack of expression. Clustering expression levels of miR-302 validated target genes showed a significant correlation between miR-302/367 cluster miRNAs and a subset of validated gene targets in healthy and adjacent tumor tissues. Taken together, our data show a highly conserved and still evolving miRNA cluster that may have additional unrecognized functions. PMID:26363379

  17. Determining the roles of a conserved tyrosine residue in a Mip-like peptidyl-prolyl cis-trans isomerase.

    PubMed

    Polley, Soumitra; Chakravarty, Devlina; Chakrabarti, Gopal; Sau, Subrata

    2016-06-01

    The FKBP22 and the related peptidyl-prolyl cis-trans isomerases dimerize using their N-terminal domains. Conversely, their C-terminal domains possess both the substrate and inhibitor binding sites. To delineate the roles of a conserved Tyr residue at their N-terminal domains, we have studied a FKBP22 mutant that carries an Ala in place of the conserved Tyr at position 15. We have demonstrated that the Tyr 15 of FKBP22 is indispensable for preserving its dimerization ability, catalytic activity, and structure. The residue, however, little contributed to its inhibitor binding ability and stability. The mode of action of Tyr 15 has been discussed at length. PMID:26944658

  18. Minimal promoter systems reveal the importance of conserved residues in the B-finger of human transcription factor IIB.

    PubMed

    Thompson, Nancy E; Glaser, Bryan T; Foley, Katherine M; Burton, Zachary F; Burgess, Richard R

    2009-09-11

    The "B-finger" of transcription factor IIB (TFIIB) is highly conserved and believed to play a role in the initiation process. We performed alanine substitutions across the B-finger of human TFIIB, made change-of-charge mutations in selected residues, and substituted the B-finger sequence from other organisms. Mutant proteins were examined in two minimal promoter systems (containing only RNA polymerase II, TATA-binding protein, and TFIIB) and in a complex system, using TFIIB-immunodepleted HeLa cell nuclear extract (NE). Mutations in conserved residues located on the sides of the B-finger had the greatest effect on activity in both minimal promoter systems, with mutations in residues Glu-51 and Arg-66 eliminating activity. The double change-of-charge mutant (E51R:R66E) did not show activity in either minimal promoter system. Mutations in the nonconserved residues at the tip of the B-finger did not significantly affect activity. However, all of the mutations in the B-finger showed at least 25% activity in the HeLa cell NE. Chimeric proteins, containing B-finger sequences from species with conserved residues on the side of the B-finger, showed wild-type activity in a minimal promoter system and in the HeLa cell NE. However, chimeric proteins whose sequence showed divergence on the sides of the B-finger had reduced activity. Transcription factor IIF (TFIIF) partially restored activity of the inactive mutants in the minimal promoter system, suggesting that TFIIF in HeLa cell NE helps to rescue the inactive mutations by interacting with either the B-finger or another component of the initiation complex that is influenced by the B-finger. PMID:19590095

  19. Conserved cysteine residues in the mammalian lamin A tail are essential for cellular responses to ROS generation.

    PubMed

    Pekovic, Vanja; Gibbs-Seymour, Ian; Markiewicz, Ewa; Alzoghaibi, Fahad; Benham, Adam M; Edwards, Robert; Wenhert, Manfred; von Zglinicki, Thomas; Hutchison, Christopher J

    2011-12-01

    Pre-lamin A and progerin have been implicated in normal aging, and the pathogenesis of age-related degenerative diseases is termed 'laminopathies'. Here, we show that mature lamin A has an essential role in cellular fitness and that oxidative damage to lamin A is involved in cellular senescence. Primary human dermal fibroblasts (HDFs) aged replicatively or by pro-oxidants acquire a range of dysmorphic nuclear shapes. We observed that conserved cysteine residues in the lamin A tail domain become hyperoxidized in senescent fibroblasts, which inhibits the formation of lamin A inter- and intramolecular disulfide bonds. Both in the absence of lamin A and in the presence of a lamin A cysteine-to-alanine mutant, which eliminates these cysteine residues (522, 588, and 591), mild oxidative stress induced nuclear disorganization and led to premature senescence as a result of decreased tolerance to ROS stimulators. Human dermal fibroblasts lacking lamin A or expressing the lamin A cysteine-to-alanine mutant displayed a gene expression profile of ROS-responsive genes characteristic of chronic ROS stimulation. Our findings suggest that the conserved C-terminal cysteine residues are essential for lamin A function and that loss or oxidative damage to these cysteine residues promotes cellular senescence. PMID:21951640

  20. Conserved residues in Lassa fever virus Z protein modulate viral infectivity at the level of the ribonucleoprotein.

    PubMed

    Capul, Althea A; de la Torre, Juan Carlos; Buchmeier, Michael J

    2011-04-01

    Arenaviruses are negative-strand RNA viruses that cause human diseases such as lymphocytic choriomeningitis, Bolivian hemorrhagic fever, and Lassa hemorrhagic fever. No licensed vaccines exist, and current treatment is limited to ribavirin. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a model for dissecting virus-host interactions in persistent and acute disease. The RING finger protein Z has been identified as the driving force of arenaviral budding and acts as the viral matrix protein. While residues in Z required for viral budding have been described, residues that govern the Z matrix function(s) have yet to be fully elucidated. Because this matrix function is integral to viral assembly, we reasoned that this would be reflected in sequence conservation. Using sequence alignment, we identified several conserved residues in Z outside the RING and late domains. Nine residues were each mutated to alanine in Lassa fever virus Z. All of the mutations affected the expression of an LCMV minigenome and the infectivity of virus-like particles, but to greatly varying degrees. Interestingly, no mutations appeared to affect Z-mediated budding or association with viral GP. Our findings provide direct experimental evidence supporting a role for Z in the modulation of the activity of the viral ribonucleoprotein (RNP) complex and its packaging into mature infectious viral particles. PMID:21228230

  1. Conserved Residues in Lassa Fever Virus Z Protein Modulate Viral Infectivity at the Level of the Ribonucleoprotein▿

    PubMed Central

    Capul, Althea A.; de la Torre, Juan Carlos; Buchmeier, Michael J.

    2011-01-01

    Arenaviruses are negative-strand RNA viruses that cause human diseases such as lymphocytic choriomeningitis, Bolivian hemorrhagic fever, and Lassa hemorrhagic fever. No licensed vaccines exist, and current treatment is limited to ribavirin. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a model for dissecting virus-host interactions in persistent and acute disease. The RING finger protein Z has been identified as the driving force of arenaviral budding and acts as the viral matrix protein. While residues in Z required for viral budding have been described, residues that govern the Z matrix function(s) have yet to be fully elucidated. Because this matrix function is integral to viral assembly, we reasoned that this would be reflected in sequence conservation. Using sequence alignment, we identified several conserved residues in Z outside the RING and late domains. Nine residues were each mutated to alanine in Lassa fever virus Z. All of the mutations affected the expression of an LCMV minigenome and the infectivity of virus-like particles, but to greatly varying degrees. Interestingly, no mutations appeared to affect Z-mediated budding or association with viral GP. Our findings provide direct experimental evidence supporting a role for Z in the modulation of the activity of the viral ribonucleoprotein (RNP) complex and its packaging into mature infectious viral particles. PMID:21228230

  2. Nitrogen and carbon mineralization from peanut residues under conservation and conventional tillage at two locations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Residue management is an important aspect of crop production systems. Availability of plant residue nitrogen (N) to succeeding crops is dependent on N mineralization rates and therefore on rates of N release during decomposition. Much of the information available on N release rates from peanut (Ar...

  3. A multi-task graph-clustering approach for chromosome conformation capture data sets identifies conserved modules of chromosomal interactions.

    PubMed

    Fotuhi Siahpirani, Alireza; Ay, Ferhat; Roy, Sushmita

    2016-01-01

    Chromosome conformation capture methods are being increasingly used to study three-dimensional genome architecture in multiple cell types and species. An important challenge is to examine changes in three-dimensional architecture across cell types and species. We present Arboretum-Hi-C, a multi-task spectral clustering method, to identify common and context-specific aspects of genome architecture. Compared to standard clustering, Arboretum-Hi-C produced more biologically consistent patterns of conservation. Most clusters are conserved and enriched for either high- or low-activity genomic signals. Most genomic regions diverge between clusters with similar chromatin state except for a few that are associated with lamina-associated domains and open chromatin. PMID:27233632

  4. Eubacterial arylamine N-acetyltransferases - identification and comparison of 18 members of the protein family with conserved active site cysteine, histidine and aspartate residues.

    PubMed

    Payton, M; Mushtaq, A; Yu, T W; Wu, L J; Sinclair, J; Sim, E

    2001-05-01

    Arylamine N-acetyltransferases (NATs) are enzymes involved in the detoxification of a range of arylamine and hydrazine-based xenobiotics. NATs have been implicated in the endogenous metabolism of p-aminobenzoyl glutamate in eukaryotes, although very little is known about the distribution and function of NAT in the prokaryotic kingdom. Using DNA library screening techniques and the analysis of data from whole-genome sequencing projects, we have identified 18 nat-like sequences from the Proteobacteria and Firmicutes. Recently, the three-dimensional structure of NAT derived from the bacterium Salmonella typhimurium (PDB accession code 1E2T) was resolved and revealed an active site catalytic triad composed of Cys(69)-His(107)-Asp(122). These residues have been shown to be conserved in all prokaryotic and eukaryotic NAT homologues together with three highly conserved regions which are found proximal to the active site triad. The characterization of prokaryotic NATs and NAT-like enzymes is reported. It is also predicted that prokaryotic NATs, based on gene cluster composition and distribution amongst genomes, participate in the metabolism of xenobiotics derived from decomposition of organic materials. PMID:11320117

  5. A Large Gene Cluster Encoding Several Magnetosome Proteins Is Conserved in Different Species of Magnetotactic Bacteria

    PubMed Central

    Grünberg, Karen; Wawer, Cathrin; Tebo, Bradley M.; Schüler, Dirk

    2001-01-01

    In magnetotactic bacteria, a number of specific proteins are associated with the magnetosome membrane (MM) and may have a crucial role in magnetite biomineralization. We have cloned and sequenced the genes of several of these polypeptides in the magnetotactic bacterium Magnetospirillum gryphiswaldense that could be assigned to two different genomic regions. Except for mamA, none of these genes have been previously reported to be related to magnetosome formation. Homologous genes were found in the genome sequences of M. magnetotacticum and magnetic coccus strain MC-1. The MM proteins identified display homology to tetratricopeptide repeat proteins (MamA), cation diffusion facilitators (MamB), and HtrA-like serine proteases (MamE) or bear no similarity to known proteins (MamC and MamD). A major gene cluster containing several magnetosome genes (including mamA and mamB) was found to be conserved in all three of the strains investigated. The mamAB cluster also contains additional genes that have no known homologs in any nonmagnetic organism, suggesting a specific role in magnetosome formation. PMID:11571158

  6. Computer-assisted re-design of spectrin SH3 residue clusters.

    PubMed

    Angrand, I; Serrano, L; Lacroix, E

    2001-10-15

    We have developed a protein design computer program, called Perla, which performs searches in sequence space to uncover optimal amino acid sequences for desired protein three-dimensional structures. Optimal sequences are localised at the minima of a sequence-structure energy landscape defined using a complex scoring function (an all-atom molecular mechanics force field plus statistical terms including entropy and solvation) measured with respect to a reference state simulating a denatured protein. Sequence choices eventually optimise side chain packing, secondary structure propensities, and hydrogen bonding and electrostatics interactions. Perla was used to re-design clusters of residues of the SH3 domain of alpha-spectrin. Several mutant proteins were produced and characterised. Some of our designed proteins have significantly higher stabilities (stability enhancements about 0.25, 0.70 and 1.0 kcal mol(-1)) than the wild-type protein. These successful protein re-designs, and similar examples found in the literature, establish the quality of the structure-based computational approach to protein design. PMID:11566604

  7. Evaluation of a high-residue cultivator for palmer amaranth control in conservation -tillage systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Resistant Palmer amaranth control in conservation systems continues to challenge producers. Recommendations currently include sequential soil applied herbicides in an attempt to prevent Palmer amaranth emergence. However, in the event activation is inadequate, alternative postemergence control opt...

  8. Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

    PubMed

    Lavrukhin, O V; Lloyd, R S

    2000-12-12

    Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates. PMID:11106507

  9. Comparative losses of glyphosate and selected residual herbicides in surface runoff from conservation-tilled watersheds planted with corn or soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conservation tillage is frequently used for corn (Zea mays L.) and soybean [Glycine max (L.) Merr] production to reduce soil loss and improve soil quality. The residual herbicides regularly used in conjunction with conservation tillage to produce these crops, however, are often detected in surface w...

  10. Carbon and nitrogen mineralization and persistence of organic residues under conservation and conventional tillage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A combination of high biomass cover crops with organic mulches may be an option for no-till vegetable production, but mineralization rates from these residues is lacking. The objective of this study was to assess nutrient release rates and persistence from mimosa, lespedeza, oat straw, and soybean r...

  11. Crop residue is key for sustaining maximum food production and for conservation of our biosphere

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Crop residue is key in our efforts to move towards agricultural sustainability. This paper provides a quick overview of some selected references and looks at some of the newest advances related to cover crops. Several authors have described in detail the benefits derived from improving soil quality ...

  12. Residue management increases fallow water conservation and yield deficit irrigated crops grown in rotation with wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    No-tillage (NT) residue management provides cover to increase precipitation capture compared with disk tillage (DT) or in the absence of a cover crop. Therefore, NT has the potential to reduce irrigation withdrawals from the declining Ogallala Aquifer. In a 4-year study, we quantified DT and NT effe...

  13. Mutation of conserved active-site threonine residues in creatine kinase affects autophosphorylation and enzyme kinetics.

    PubMed Central

    Stolz, Martin; Hornemann, Thorsten; Schlattner, Uwe; Wallimann, Theo

    2002-01-01

    Muscle-type creatine kinase (MM-CK) is a member of an isoenzyme family with key functions in cellular energetics. It has become a matter of debate whether the enzyme is autophosphorylated, as reported earlier [Hemmer, Furter-Graves, Frank, Wallimann and Furter (1995) Biochim. Biophys. Acta 1251, 81-90], or exclusively nucleotidylated. In the present paper, we demonstrate unambiguously that CK is indeed autophosphorylated. However, this autophosphorylation is not solely responsible for the observed microheterogeneity of MM-CK on two-dimensional isoelectric focusing gels. Using phosphoamino-acid analysis of (32)P-labelled CK isoforms, phosphothreonine (P-Thr) residues were identified as the only product of autophosphorylation for all CK isoenzymes. The phosphorylated residues in chicken MM-CK were allocated to a region in the vicinity of the active site, where five putative phosphorylation sites were identified. Site-directed threonine-valine-replacement mutants reveal that autophosphorylation is not specific for one particular residue but occurs at all examined threonine residues. The enzyme kinetic parameters indicate that the autophosphorylation of CK exerts a modulatory effect on substrate binding and the equilibrium constant, rather than on the catalytic mechanism itself. PMID:11964180

  14. Conservation.

    ERIC Educational Resources Information Center

    National Audubon Society, New York, NY.

    This set of teaching aids consists of seven Audubon Nature Bulletins, providing the teacher and student with informational reading on various topics in conservation. The bulletins have these titles: Plants as Makers of Soil, Water Pollution Control, The Ground Water Table, Conservation--To Keep This Earth Habitable, Our Threatened Air Supply,…

  15. Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins

    PubMed Central

    Tanaka, Motoko; Robinson, Bridget A.; Chutiraka, Kasana; Geary, Clair D.; Reed, Jonathan C.

    2015-01-01

    ABSTRACT The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer. IMPORTANCE The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly

  16. Two conserved arginine residues from the SK3 potassium channel outer vestibule control selectivity of recognition by scorpion toxins.

    PubMed

    Feng, Jing; Hu, Youtian; Yi, Hong; Yin, Shijin; Han, Song; Hu, Jun; Chen, Zongyun; Yang, Weishan; Cao, Zhijian; De Waard, Michel; Sabatier, Jean-Marc; Li, Wenxin; Wu, Yingliang

    2013-05-01

    Potassium channel functions are often deciphered by using selective and potent scorpion toxins. Among these toxins, only a limited subset is capable of selectively blocking small conductance Ca(2+)-activated K(+) (SK) channels. The structural bases of this selective SK channel recognition remain unclear. In this work, we demonstrate the key role of the electric charges of two conserved arginine residues (Arg-485 and Arg-489) from the SK3 channel outer vestibule in the selective recognition by the SK3-blocking BmP05 toxin. Indeed, individually substituting these residues with histidyl or lysyl (maintaining the positive electric charge partially or fully), although decreasing BmP05 affinity, still preserved the toxin sensitivity profile of the SK3 channel (as evidenced by the lack of recognition by many other types of potassium channel-sensitive charybdotoxin). In contrast, when Arg-485 or Arg-489 of the SK3 channel was mutated to an acidic (Glu) or alcoholic (Ser) amino acid residue, the channel lost its sensitivity to BmP05 and became susceptible to the "new" blocking activity by charybdotoxin. In addition to these SK3 channel basic residues important for sensitivity, two acidic residues, Asp-492 and Asp-518, also located in the SK3 channel outer vestibule, were identified as being critical for toxin affinity. Furthermore, molecular modeling data indicate the existence of a compact SK3 channel turret conformation (like a peptide screener), where the basic rings of Arg-485 and Arg-489 are stabilized by strong ionic interactions with Asp-492 and Asp-518. In conclusion, the unique properties of Arg-485 and Arg-489 (spatial orientations and molecular interactions) in the SK3 channel account for its toxin sensitivity profile. PMID:23511633

  17. Integrating herbicides in a high-residue cover crop conservation agriculture setting

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conservation agriculture systems provide a means to ensure long-term agricultural productivity, protect environmental quality, and reduce inputs into farming systems. Weed control in these systems rely on multiple tactics to achieve effective weed management while limiting chemical inputs. Practic...

  18. Fall conservation deep tillage stabilizes maize residues into soil organic matter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Efforts for increasing soil organic matter (SOM) content under agricultural systems have primarily focused on management practices that reduce exposure of SOM to decomposition via minimum tillage. We assess an alternative approach, termed ‘fall conservation deep tillage’ (FCDT), to SOM stabilization...

  19. WEED CONTROL IN CONSERVATION TILLAGE TOMATOES FOLLOWING HERBICIDE AND COVER CROP RESIDUE INTEGRATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An experiment was conducted in 2005 and 2006 at the North Alabama Horticulture Experiment Station located in Cullman, AL. A similar experiment was conducted in 2006 at George Washington Carver Agricultural Experiment Station of Tuskegee University at Tuskegee, AL. Three conservation-tillage systems ...

  20. Evolution of conserved non-coding sequences within the vertebrate Hox clusters through the two-round whole genome duplications revealed by phylogenetic footprinting analysis.

    PubMed

    Matsunami, Masatoshi; Sumiyama, Kenta; Saitou, Naruya

    2010-12-01

    As a result of two-round whole genome duplications, four or more paralogous Hox clusters exist in vertebrate genomes. The paralogous genes in the Hox clusters show similar expression patterns, implying shared regulatory mechanisms for expression of these genes. Previous studies partly revealed the expression mechanisms of Hox genes. However, cis-regulatory elements that control these paralogous gene expression are still poorly understood. Toward solving this problem, the authors searched conserved non-coding sequences (CNSs), which are candidates of cis-regulatory elements. When comparing orthologous Hox clusters of 19 vertebrate species, 208 intergenic conserved regions were found. The authors then searched for CNSs that were conserved not only between orthologous clusters but also among the four paralogous Hox clusters. The authors found three regions that are conserved among all the four clusters and eight regions that are conserved between intergenic regions of two paralogous Hox clusters. In total, 28 CNSs were identified in the paralogous Hox clusters, and nine of them were newly found in this study. One of these novel regions bears a RARE motif. These CNSs are candidates for gene expression regulatory regions among paralogous Hox clusters. The authors also compared vertebrate CNSs with amphioxus CNSs within the Hox cluster, and found that two CNSs in the HoxA and HoxB clusters retain homology with amphioxus CNSs through the two-round whole genome duplications. PMID:20981416

  1. Studies on the fusion peptide of a paramyxovirus fusion glycoprotein: roles of conserved residues in cell fusion.

    PubMed Central

    Horvath, C M; Lamb, R A

    1992-01-01

    The role of residues in the conserved hydrophobic N-terminal fusion peptide of the paramyxovirus fusion (F) protein in causing cell-cell fusion was examined. Mutations were introduced into the cDNA encoding the simian virus 5 (SV5) F protein, the altered F proteins were expressed by using an eukaryotic vector, and their ability to mediate syncytium formation was determined. The mutant F proteins contained both single- and multiple-amino-acid substitutions, and they exhibited a variety of intracellular transport properties and fusion phenotypes. The data indicate that many substitutions in the conserved amino acids of the simian virus 5 F fusion peptide can be tolerated without loss of biological activity. Mutant F proteins which were not transported to the cell surface did not cause cell-cell fusion, but all of the mutants which were transported to the cell surface were fusion competent, exhibiting fusion properties similar to or better than those of the wild-type F protein. Mutant F proteins containing glycine-to-alanine substitutions had altered intracellular transport characteristics, yet they exhibited a great increase in fusion activity. The potential structural implications of this substitution and the possible importance of these glycine residues in maintaining appropriate levels of fusion activity are discussed. Images PMID:1548771

  2. A Conserved Acidic Residue in Phenylalanine Hydroxylase Contributes to Cofactor Affinity and Catalysis

    PubMed Central

    2015-01-01

    The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of

  3. Role of conserved Met112 residue in the catalytic activity and stability of ketosteroid isomerase.

    PubMed

    Cha, Hyung Jin; Jang, Do Soo; Jeong, Jae-Hee; Hong, Bee Hak; Yun, Young Sung; Shin, Eun Ju; Choi, Kwan Yong

    2016-10-01

    Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core. PMID:27375051

  4. Identification of highly conserved residues involved in inhibition of HIV-1 RNase H function by Diketo acid derivatives.

    PubMed

    Corona, Angela; Di Leva, Francesco Saverio; Thierry, Sylvain; Pescatori, Luca; Cuzzucoli Crucitti, Giuliana; Subra, Frederic; Delelis, Olivier; Esposito, Francesca; Rigogliuso, Giuseppe; Costi, Roberta; Cosconati, Sandro; Novellino, Ettore; Di Santo, Roberto; Tramontano, Enzo

    2014-10-01

    HIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg(2+)-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors. PMID:25092689

  5. Analysis of Conserved Glutamate and Aspartate Residues in Drosophila Rhodopsin 1 and Their Influence on Spectral Tuning.

    PubMed

    Zheng, Lijun; Farrell, David M; Fulton, Ruth M; Bagg, Eve E; Salcedo, Ernesto; Manino, Meridee; Britt, Steven G

    2015-09-01

    The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Studies of amphioxus Go-opsin have demonstrated that Glu-181 functions as the counterion in this pigment. This finding has led to the proposal that Glu-181 may function as the counterion in other invertebrate visual pigments as well. Here we describe a series of mutagenesis experiments to test this hypothesis and to also test whether other conserved acidic amino acids in Drosophila Rhodopsin 1 (Rh1) may serve as the counterion of this visual pigment. Of the 5 Glu and Asp residues replaced by Gln or Asn in our experiments, none of the mutant pigments shift the absorption of Rh1 by more than 6 nm. In combination with prior studies, these results suggest that the counterion in Drosophila Rh1 may not be located at Glu-181 as in amphioxus, or at Glu-113 as in bovine rhodopsin. Conversely, the extremely low steady state levels of the E194Q mutant pigment (bovine opsin site Glu-181), and the rhabdomere degeneration observed in flies expressing this mutant demonstrate that a negatively charged residue at this position is essential for normal rhodopsin function in vivo. This work also raises the possibility that another residue or physiologic anion may compensate for the missing counterion in the E194Q mutant. PMID:26195627

  6. Topologically Conserved Residues Direct Heme Transport in HRG-1-related Proteins*

    PubMed Central

    Yuan, Xiaojing; Protchenko, Olga; Philpott, Caroline C.; Hamza, Iqbal

    2012-01-01

    Caenorhabditis elegans and human HRG-1-related proteins are conserved, membrane-bound permeases that bind and translocate heme in metazoan cells via a currently uncharacterized mechanism. Here, we show that cellular import of heme by HRG-1-related proteins from worms and humans requires strategically located amino acids that are topologically conserved across species. We exploit a heme synthesis-defective Saccharomyces cerevisiae mutant to model the heme auxotrophy of C. elegans and demonstrate that, under heme-deplete conditions, the endosomal CeHRG-1 requires both a specific histidine in the predicted second transmembrane domain (TMD2) and the FARKY motif in the C terminus tail for heme transport. By contrast, the plasma membrane CeHRG-4 transports heme by utilizing a histidine in the exoplasmic (E2) loop and the FARKY motif. Optimal activity under heme-limiting conditions, however, requires histidine in the E2 loop of CeHRG-1 and tyrosine in TMD2 of CeHRG-4. An analogous system exists in humans, because mutation of the synonymous histidine in TMD2 of hHRG-1 eliminates heme transport activity, implying an evolutionary conserved heme transport mechanism that predates vertebrate origins. Our results support a model in which heme is translocated across membranes facilitated by conserved amino acids positioned on the exoplasmic, cytoplasmic, and transmembrane regions of HRG-1-related proteins. These findings may provide a framework for understanding the structural basis of heme transport in eukaryotes and human parasites, which rely on host heme for survival. PMID:22174408

  7. RESIDUAL GAS MOTIONS IN THE INTRACLUSTER MEDIUM AND BIAS IN HYDROSTATIC MEASUREMENTS OF MASS PROFILES OF CLUSTERS

    SciTech Connect

    Lau, Erwin T.; Kravtsov, Andrey V.; Nagai, Daisuke

    2009-11-10

    We present analysis of bulk and random gas motions in the intracluster medium using high-resolution Eulerian cosmological simulations of 16 simulated clusters, including both very relaxed and unrelaxed systems and spanning a virial mass range of 5 x 10{sup 13} - 2 x 10{sup 15} h{sup -1} M-odot. We investigate effects of the residual subsonic gas motions on the hydrostatic estimates of mass profiles and concentrations of galaxy clusters. In agreement with previous studies, we find that the gas motions contribute up to approx5%-15% of the total pressure support in relaxed clusters with contribution increasing with the cluster-centric radius. The fractional pressure support is higher in unrelaxed systems. This contribution would not be accounted for in hydrostatic estimates of the total mass profile and would lead to systematic underestimate of mass. We demonstrate that total mass can be recovered accurately if pressure due to gas motions measured in simulations is explicitly taken into account in the equation of hydrostatic equilibrium. Given that the underestimate of mass is increasing at larger radii, where gas is less relaxed and contribution of gas motions to pressure is larger, the total density profile derived from hydrostatic analysis is more concentrated than the true profile. This may at least partially explain some high values of concentrations of clusters estimated from hydrostatic analysis of X-ray data.

  8. The Conserved Arginine Cluster in the Insert of the Third Cytoplasmic Loop of the Long Form of the D2 Dopamine Receptor (D2L-R) Acts as an Intracellular Retention Signal

    PubMed Central

    Kubale, Valentina; Blagotinšek, Kaja; Nøhr, Jane; Eidne, Karin A.; Vrecl, Milka

    2016-01-01

    This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D2 dopamine receptor (D2L-R) confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR) located within the insert could act as an RXR-type endoplasmic reticulum (ER) retention signal. Arginine residues (R) within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E), either individually or in clusters, thus generating single, double, and triple D2L-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA), radioligand binding assay, bioluminescence resonance energy transfer (BRET2) β-arrestin 2 (βarr2) recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E) substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D2L-R mutants preserved their functional integrity regarding ligand binding, agonist-induced βarr2 recruitment and Gαi-mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3) of the D2L-R appears to be the ER retention signal. PMID:27447620

  9. Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY.

    PubMed Central

    Bourret, R B; Hess, J F; Simon, M I

    1990-01-01

    The CheY protein is phosphorylated by CheA and dephosphorylated by CheZ as part of the chemotactic signal transduction pathway in Escherichia coli. Phosphorylation of CheY has been proposed to occur on an aspartate residue. Each of the eight aspartate residues of CheY was replaced by using site-directed mutagenesis. Substitutions at Asp-12, Asp-13, or Asp-57 resulted in loss of chemotaxis. Most of the mutant CheY proteins were still phosphorylated by CheA but exhibited modified biochemical properties, including reduced ability to accept phosphate from CheA, altered phosphate group stability, and/or resistance to CheZ-mediated dephosphorylation. The properties of CheY proteins bearing a substitution at position 57 were most aberrant, consistent with the hypothesis that Asp-57 is the normal site of acyl phosphate formation. Evidence for an alternate site of phosphorylation in the Asp-57 mutants is presented. Phosphorylated CheY is believed to cause tumbling behavior. However, a dominant mutant CheY protein that was not phosphorylated in vitro caused tumbling in vivo in the absence of CheA. This phenotype suggests that the role of phosphorylation in the wild-type CheY protein is to stabilize a transient conformational change that can generate tumbling behavior. Images PMID:2404281

  10. Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue

    DOE PAGESBeta

    Teixeira, Miguel; Cabelli, Diane; Pinto, Ana F.; Romao, Celia V.; Pinto, Liliana C.; Huber, Harald; Saraiva, Ligia M.; Todorovic, Smilja

    2014-12-05

    Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the –EKHVP– motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue ismore » substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (₋E₂₃T₂₄HVP₋), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.« less

  11. Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue

    SciTech Connect

    Teixeira, Miguel; Cabelli, Diane; Pinto, Ana F.; Romao, Celia V.; Pinto, Liliana C.; Huber, Harald; Saraiva, Ligia M.; Todorovic, Smilja

    2014-12-05

    Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the –EKHVP– motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue is substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (₋E₂₃T₂₄HVP₋), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.

  12. Analysis of Conserved Glutamate and Aspartate Residues in Drosophila Rhodopsin 1 and Their Influence on Spectral Tuning*

    PubMed Central

    Zheng, Lijun; Farrell, David M.; Fulton, Ruth M.; Bagg, Eve E.; Salcedo, Ernesto; Manino, Meridee; Britt, Steven G.

    2015-01-01

    The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Studies of amphioxus Go-opsin have demonstrated that Glu-181 functions as the counterion in this pigment. This finding has led to the proposal that Glu-181 may function as the counterion in other invertebrate visual pigments as well. Here we describe a series of mutagenesis experiments to test this hypothesis and to also test whether other conserved acidic amino acids in Drosophila Rhodopsin 1 (Rh1) may serve as the counterion of this visual pigment. Of the 5 Glu and Asp residues replaced by Gln or Asn in our experiments, none of the mutant pigments shift the absorption of Rh1 by more than 6 nm. In combination with prior studies, these results suggest that the counterion in Drosophila Rh1 may not be located at Glu-181 as in amphioxus, or at Glu-113 as in bovine rhodopsin. Conversely, the extremely low steady state levels of the E194Q mutant pigment (bovine opsin site Glu-181), and the rhabdomere degeneration observed in flies expressing this mutant demonstrate that a negatively charged residueat this position is essential for normal rhodopsin function in vivo. This work also raises the possibility that another residue or physiologic anion may compensate for the missing counterion in the E194Q mutant. PMID:26195627

  13. Density functional theory calculations on the active site of biotin synthase: mechanism of S transfer from the Fe(2)S(2) cluster and the role of 1st and 2nd sphere residues.

    PubMed

    Rana, Atanu; Dey, Subal; Agrawal, Amita; Dey, Abhishek

    2015-10-01

    Density functional theory (DFT) calculations are performed on the active site of biotin synthase (BS) to investigate the sulfur transfer from the Fe(2)S(2) cluster to dethiobiotin (DTB). The active site is modeled to include both the 1st and 2nd sphere residues. Molecular orbital theory considerations and calculation on smaller models indicate that only an S atom (not S²⁻) transfer from an oxidized Fe(2)S(2) cluster leads to the formation of biotin from the DTB using two adenosyl radicals generated from S-adenosyl-L-methionine. The calculations on larger protein active site model indicate that a 9-monothiobiotin bound reduced cluster should be an intermediate during the S atom insertion from the Fe(2)S(2) cluster consistent with experimental data. The Arg260 bound to Fe1, being a weaker donor than cysteine bound to Fe(2), determines the geometry and the electronic structure of this intermediate. The formation of this intermediate containing the C9-S bond is estimated to have a ΔG(≠) of 17.1 kcal/mol while its decay by the formation of the 2nd C6-S bond is calculated to have a ΔG(≠) of 29.8 kcal/mol, i.e. the 2nd C-S bond formation is calculated to be the rate determining step in the cycle and it leads to the decay of the Fe(2)S(2) cluster. Significant configuration interaction (CI), present in these transition states, helps lower the barrier of these reactions by ~30-25 kcal/mol relative to a hypothetical outer-sphere reaction. The conserved Phe285 residue near the Fe(2)S(2) active site determines the stereo selectivity at the C6 center of this radical coupling reaction. Reaction mechanism of BS investigated using DFT calculations. Strong CI and the Phe285 residue control the kinetic rate and stereochemistry of the product. PMID:26369537

  14. The Role of Conserved Tyrosine Residues in NiSOD Catalysis: A Case of Convergent Evolution

    PubMed Central

    Herbst, Robert W.; Guce, Abigail; Bryngelson, Peter A.; Higgins, Khadine A.; Ryan, Kelly C.; Cabelli, Diane E.; Garman, Scott C.; Maroney, Michael J.

    2013-01-01

    Superoxide dismutases rely on protein structural elements to adjust the redox potential of the metallocenter to an optimum value near 300 mV (vs. NHE), to provide a source of protons for catalysis, and to control the access of anions to the active site. These aspects of the catalytic mechanism are examined herein for recombinant preparations of the nickel-dependent SOD (NiSOD) from Streptomyces coelicolor, and for a series of mutants that affect a key tyrosine residue, Tyr9 (Y9F-, Y62F-, Y9FY62F- and D3A-NiSOD). Structural aspects of the nickel sites are examined by a combination of EPR and x-ray absorption spectroscopies, and by single crystal x-ray diffraction at ~ 1.9 Å resolution in the case of Y9F- and D3A-NiSODs. The functional effects of the mutations are examined by kinetic studies employing pulse radiolytic generation of O2− and by redox titrations. These studies reveal that although the structure of the nickel center in NiSOD is unique, the ligand environment is designed to optimize the redox potential at 290 mV and results in the oxidation of 50% of the nickel centers in the oxidized hexamer. Kinetic investigations show that all of the mutant proteins have considerable activity. In the case of Y9F-NiSOD, the enzyme shows saturation behavior that is not observed in WT-NiSOD and suggests that release of peroxide is inhibited. The crystal structure of Y9F-NiSOD reveals an anion binding site that is occupied by either Cl− or Br− and is located close to, but not within bonding distance of the nickel center. The structure of D3A-NiSOD reveals that in addition to affecting the interaction between subunits, this mutation repositions Y9 and leads to altered chemistry with peroxide. Comparisons with Mn(SOD) and Fe(SOD) reveal that although different strategies are employed to adjust the redox potential and supply of protons, NiSOD has evolved a similar strategy to control the access of anions to the active site. PMID:19183068

  15. Molecular Dynamics simulations of the electrospray process: formation of NaCl clusters via the charged residue mechanism.

    PubMed

    Konermann, Lars; McAllister, Robert G; Metwally, Haidy

    2014-10-16

    Electrospray ionization (ESI) produces desolvated ions from solution phase analytes for mass spectrometric detection. The final steps of gas phase ion formation from nanometer-sized solvent droplets remain a matter of debate. According to the ion evaporation model (IEM), analytes are ejected from the droplet surface via field emission, whereas the charged residue model (CRM) envisions that ions are released upon droplet evaporation to dryness. Exposure of salt solutions to ESI conditions produces a range of cluster ions. Despite the rich literature on these systems, it is still unclear if these salt clusters form via the CRM or the IEM. The current study explores the formation of Na(n)Cl(m)((n-m)+) clusters from aqueous sodium chloride solution under positive and negative polarity conditions. Molecular dynamics (MD) methods are used for simulating the temporal evolution of charged NaCl-containing water droplets. A trajectory stitching approach is developed for continuously removing evaporated moieties from the simulation, thereby dramatically reducing computational cost. In addition, this procedure ensures adequate temperature control and eliminates evaporative cooling that would otherwise slow down the process. Continuous water evaporation leads to progressive droplet shrinkage, while the emission of solvated single ions ensures that the system remains at ca. 90% of the Rayleigh limit. Early during the process all ions in the droplet behave as freely dissolved species, but after a few nanoseconds at 370 K the systems gradually morph into amorphous wet salt aggregates. Ultimately, free Na(n)Cl(m)((n-m)+) clusters form as the last solvent molecules evaporate. Our data therefore provide direct evidence that sodium chloride cluster formation during ESI proceeds via the CRM. The IEM nonetheless plays an ancillary role, as it allows the system to shed charge (mostly in the form of hydrated Na(+) or Cl(-)) during droplet shrinkage. It appears that this study marks the

  16. Conserved piRNA Expression from a Distinct Set of piRNA Cluster Loci in Eutherian Mammals

    PubMed Central

    Zeng, Mei; Gerlach, Daniel; Yu, Michael; Berger, Bonnie; Naramura, Mayumi; Kile, Benjamin T.; Lau, Nelson C.

    2015-01-01

    The Piwi pathway is deeply conserved amongst animals because one of its essential functions is to repress transposons. However, many Piwi-interacting RNAs (piRNAs) do not base-pair to transposons and remain mysterious in their targeting function. The sheer number of piRNA cluster (piC) loci in animal genomes and infrequent piRNA sequence conservation also present challenges in determining which piC loci are most important for development. To address this question, we determined the piRNA expression patterns of piC loci across a wide phylogenetic spectrum of animals, and reveal that most genic and intergenic piC loci evolve rapidly in their capacity to generate piRNAs, regardless of known transposon silencing function. Surprisingly, we also uncovered a distinct set of piC loci with piRNA expression conserved deeply in Eutherian mammals. We name these loci Eutherian-Conserved piRNA cluster (ECpiC) loci. Supporting the hypothesis that conservation of piRNA expression across ~100 million years of Eutherian evolution implies function, we determined that one ECpiC locus generates abundant piRNAs antisense to the STOX1 transcript, a gene clinically associated with preeclampsia. Furthermore, we confirmed reduced piRNAs in existing mouse mutations at ECpiC-Asb1 and -Cbl, which also display spermatogenic defects. The Asb1 mutant testes with strongly reduced Asb1 piRNAs also exhibit up-regulated gene expression profiles. These data indicate ECpiC loci may be specially adapted to support Eutherian reproduction. PMID:26588211

  17. Production of Bioactive Diterpenoids in the Euphorbiaceae Depends on Evolutionarily Conserved Gene Clusters[C][W][OPEN

    PubMed Central

    King, Andrew J.; Brown, Geoffrey D.; Gilday, Alison D.; Larson, Tony R.; Graham, Ian A.

    2014-01-01

    The Euphorbiaceae produce a diverse range of diterpenoids, many of which have pharmacological activities. These diterpenoids include ingenol mebutate, which is licensed for the treatment of a precancerous skin condition (actinic keratosis), and phorbol derivatives such as resiniferatoxin and prostratin, which are undergoing investigation for the treatment of severe pain and HIV, respectively. Despite the interest in these diterpenoids, their biosynthesis is poorly understood at present, with the only characterized step being the conversion of geranylgeranyl pyrophosphate into casbene. Here, we report a physical cluster of diterpenoid biosynthetic genes from castor (Ricinus communis), including casbene synthases and cytochrome P450s from the CYP726A subfamily. CYP726A14, CYP726A17, and CYP726A18 were able to catalyze 5-oxidation of casbene, a conserved oxidation step in the biosynthesis of this family of medicinally important diterpenoids. CYP726A16 catalyzed 7,8-epoxidation of 5-keto-casbene and CYP726A15 catalyzed 5-oxidation of neocembrene. Evidence of similar gene clustering was also found in two other Euphorbiaceae, including Euphorbia peplus, the source organism of ingenol mebutate. These results demonstrate conservation of gene clusters at the higher taxonomic level of the plant family and that this phenomenon could prove useful in further elucidating diterpenoid biosynthetic pathways. PMID:25172144

  18. Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA.

    PubMed

    Hara, Noritaka; Namba, Keiichi; Minamino, Tohru

    2011-01-01

    For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate. PMID:21811603

  19. A conserved interdomain communication pathway of pseudosymmetrically distributed residues affects substrate specificity of the fungal multidrug transporter Cdr1p.

    PubMed

    Kolaczkowski, Marcin; Sroda-Pomianek, Kamila; Kolaczkowska, Anna; Michalak, Krystyna

    2013-02-01

    Understanding the communication pathways between remote sites in proteins is of key importance for understanding their function and mechanism of action. These remain largely unexplored among the pleiotropic drug resistance (PDR) representatives of the ubiquitous superfamily of ATP-binding cassette (ABC) transporters. To identify functionally coupled residues important for the polyspecific transport by the fungal ABC multidrug transporter Cdr1p a new selection strategy, towards increased resistance to a preferred substrate of the homologous Snq2p, was applied to a library of randomly generated mutants. The single amino acid substitutions, located pseudosymmetrically in each domain of the internally duplicated protein: the H-loop of the N-terminal nucleotide binding domain (NBD1) (C363R) and in the C-terminal NBD2 region preceding Walker A (V885G). The central regions of the first transmembrane helices 1 and 7 of both transmembrane domains were also affected by the G521S/D and A1208V substitutions respectively. Although the mutants were expressed at a similar level and located correctly to the plasma membrane, they selectively affected transport of multiple drugs, including azole antifungals. The synergistic effects of combined mutations on drug resistance, drug dependent ATPase activity and transport support the view inferred from the statistical coupling analysis (SCA) of aminoacid coevolution and mutational analysis of other ABC transporter families that these residues are an important part of the conserved, allosterically coupled interdomain communication network. Our results shed new light on the communication between the pseudosymmetrically arranged domains in a fungal PDR ABC transporter and reveal its profound influence on substrate specificity. PMID:23122779

  20. RESIDUAL AND CONTACT HERBICIDE LOSSES IN SURFACE RUNOFF FROM CONSERVATION TILLED WATERSHEDS PLANTED WITH TRANSGENIC, HERBICIDE-TOLERANT, CORN AND SOYBEAN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conservation tillage is frequently used for corn (Zea mays L.) and soybean [Glycine max (L.) Merr] production to reduce soil loss, improve soil quality, and maintain eligibility for commodity support payments. The residual herbicides normally used in the production of these crops, however, are often...

  1. Crop residues of the contiguous United States: Balancing feedstock and soil needs with conservation tillage, cover crops, and biochar

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Crop residues are among the cellulosic feedstocks expected to provide renewable energy. The availability of crop species and residue availability varies across the United States. Estimates of harvestable residues must consider all the residues produced during the entire rotation. Inclusion of fallow...

  2. Nucleotide sequence of a cluster of early and late genes in a conserved segment of the vaccinia virus genome.

    PubMed Central

    Plucienniczak, A; Schroeder, E; Zettlmeissl, G; Streeck, R E

    1985-01-01

    The nucleotide sequence of a 7.6 kb vaccinia DNA segment from a genomic region conserved among different orthopox virus has been determined. This segment contains a tight cluster of 12 partly overlapping open reading frames most of which can be correlated with previously identified early and late proteins and mRNAs. Regulatory signals used by vaccinia virus have been studied. Presumptive promoter regions are rich in A, T and carry the consensus sequences TATA and AATAA spaced at 20-24 base pairs. Tandem repeats of a CTATTC consensus sequence are proposed to be involved in the termination of early transcription. PMID:2987815

  3. IRBIT Interacts with the Catalytic Core of Phosphatidylinositol Phosphate Kinase Type Iα and IIα through Conserved Catalytic Aspartate Residues

    PubMed Central

    Ando, Hideaki; Hirose, Matsumi; Gainche, Laura; Kawaai, Katsuhiro; Bonneau, Benjamin; Ijuin, Takeshi; Itoh, Toshiki; Takenawa, Tadaomi; Mikoshiba, Katsuhiko

    2015-01-01

    Phosphatidylinositol phosphate kinases (PIPKs) are lipid kinases that generate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a critical lipid signaling molecule that regulates diverse cellular functions, including the activities of membrane channels and transporters. IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate) is a multifunctional protein that regulates diverse target proteins. Here, we report that IRBIT forms signaling complexes with members of the PIPK family. IRBIT bound to all PIPK isoforms in heterologous expression systems and specifically interacted with PIPK type Iα (PIPKIα) and type IIα (PIPKIIα) in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are involved in the interaction with IRBIT. Furthermore, phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfered with the interaction, suggesting that IRBIT interacts with catalytic cores of PIPKs. Mutations of phosphorylation sites in the serine-rich region of IRBIT affected the selectivity of its interaction with PIPKIα and PIPKIIα. The structural flexibility of the serine-rich region, located in the intrinsically disordered protein region, is assumed to underlie the mechanism of this interaction. Furthermore, in vitro binding experiments and immunocytochemistry suggest that IRBIT and PIPKIα interact with the Na+/HCO3− cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIα and NBCe1-B, whose activity is regulated by PI(4,5)P2. PMID:26509711

  4. A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding

    PubMed Central

    2013-01-01

    Background Streptomyces sp. N174 chitosanase (CsnN174), a member of glycoside hydrolases family 46, is one of the most extensively studied chitosanases. Previous studies allowed identifying several key residues of this inverting enzyme, such as the two catalytic carboxylic amino acids as well as residues that are involved in substrate binding. In spite of the progress in understanding the catalytic mechanism of this chitosanase, the function of some residues highly conserved throughout GH46 family has not been fully elucidated. This study focuses on one of such residues, the arginine 42. Results Mutation of Arg42 into any other amino acid resulted in a drastic loss of enzyme activity. Detailed investigations of R42E and R42K chitosanases revealed that the mutant enzymes are not only impaired in their catalytic activity but also in their mode of interaction with the substrate. Mutated enzymes were more sensitive to substrate inhibition and were altered in their pattern of activity against chitosans of various degrees of deacetylation. Our data show that Arg42 plays a dual role in CsnN174 activity. Conclusions Arginine 42 is essential to maintain the enzymatic function of chitosanase CsnN174. We suggest that this arginine is influencing the catalytic nucleophile residue and also the substrate binding mode of the enzyme by optimizing the electrostatic interaction between the negatively charged carboxylic residues of the substrate binding cleft and the amino groups of GlcN residues in chitosan. PMID:24041306

  5. MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus.

    PubMed

    Chakraborty, Sandeep

    2016-01-01

    The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV), provides key insights in developing strategies for tackling ZIKV. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little emphasis in existing literature, are found to have significant electrostatic perturbation. Thus, a combination of different computational methods enable the rapid and rational detection of critical residues that can be made the target of small drugs, or as epitopes in the search for an elusive therapy or vaccine that neutralizes multiple members of the Flaviviridae family. PMID:27540468

  6. MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus

    PubMed Central

    Chakraborty, Sandeep

    2016-01-01

    The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV), provides key insights in developing strategies for tackling ZIKV. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little emphasis in existing literature, are found to have significant electrostatic perturbation. Thus, a combination of different computational methods enable the rapid and rational detection of critical residues that can be made the target of small drugs, or as epitopes in the search for an elusive therapy or vaccine that neutralizes multiple members of the Flaviviridae family. PMID:27540468

  7. The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines.

    PubMed

    Kim, Hyo-Jin; Lee, Kang-Lok; Kim, Kyoung-Dong; Roe, Jung-Hye

    2016-09-01

    Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1. PMID:27444384

  8. Functional Requirement for a Highly Conserved Charged Residue at Position 75 in the Gap Junction Protein Connexin 32*

    PubMed Central

    Abrams, Charles K.; Islam, Mahee; Mahmoud, Rola; Kwon, Taekyung; Bargiello, Thaddeus A.; Freidin, Mona M.

    2013-01-01

    Charcot Marie Tooth disease (CMT) is a group of inherited disorders characterized clinically by exclusively or predominantly peripheral nerve dysfunction. CMT1X, the most common form of X-linked CMT is caused by mutations in connexin 32 (Cx32). In this work, we used dual whole cell patch clamp recording to examine the functional effects of mutations at the Arg75 position. This residue is highly conserved among members of the connexin family, and disease-causing mutations have been identified at this (or the corresponding) position in Cx26, Cx43, and Cx46. Thus, a better understanding of the effects of mutations of this position in Cx32 may have relevance to pathogenesis of a number of different human diseases. All three mutants associated with CMT1X (R75P, R75Q, and R75W) showed very low levels of coupling similar to those of the cells transfected with vector alone. Heterotypic pairing with Cx32 WT showed that the absence of coupling for these mutants in the homotypic configuration could be explained by shifts in their hemichannel Gj-Vj relations. Examination of the expression levels and gating characteristics of seven additional mutants (R75A, R75D, R75E, R75H, R75K, R75L, and R75V) at this position suggest that the positive charge at position 75 in Cx32 is required for normal channel function but not for gap junction assembly. Our studies also suggest that disease treatment strategies for CMT1X, which correct trafficking abnormalities in Cx32, may be ineffective for the group of mutations also conferring changes in gating properties of Cx32 channels. PMID:23209285

  9. Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex.

    PubMed

    You, Zhiying; Ode, Koji L; Shindo, Mayumi; Takisawa, Haruhiko; Masai, Hisao

    2016-05-01

    All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2∼7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity. PMID:26940553

  10. Conserved Single Residue in the BK Potassium Channel Required for Activation by Alcohol and Intoxication in C. elegans

    PubMed Central

    Davis, Scott J.; Scott, Luisa L.; Hu, Kevin

    2014-01-01

    Alcohol directly modulates the BK potassium channel to alter behaviors in species ranging from invertebrates to humans. In the nematode Caenorhabditis elegans, mutations that eliminate the BK channel, SLO-1, convey dramatic resistance to intoxication by ethanol. We hypothesized that certain conserved amino acids are critical for ethanol modulation, but not for basal channel function. To identify such residues, we screened C. elegans strains with different missense mutations in the SLO-1 channel. A strain with the SLO-1 missense mutation T381I in the RCK1 domain was highly resistant to intoxication. This mutation did not interfere with other BK channel-dependent behaviors, suggesting that the mutant channel retained normal in vivo function. Knock-in of wild-type versions of the worm or human BK channel rescued intoxication and other BK channel-dependent behaviors in a slo-1-null mutant background. In contrast, knock-in of the worm T381I or equivalent human T352I mutant BK channel selectively rescued BK channel-dependent behaviors while conveying resistance to intoxication. Single-channel patch-clamp recordings confirmed that the human BK channel engineered with the T352I missense mutation was insensitive to activation by ethanol, but otherwise had normal conductance, potassium selectivity, and only subtle differences in voltage dependence. Together, our behavioral and electrophysiological results demonstrate that the T352I mutation selectively disrupts ethanol modulation of the BK channel. The T352I mutation may alter a binding site for ethanol and/or interfere with ethanol-induced conformational changes that are critical for behavioral responses to ethanol. PMID:25031399

  11. Conserved single residue in the BK potassium channel required for activation by alcohol and intoxication in C. elegans.

    PubMed

    Davis, Scott J; Scott, Luisa L; Hu, Kevin; Pierce-Shimomura, Jonathan T

    2014-07-16

    Alcohol directly modulates the BK potassium channel to alter behaviors in species ranging from invertebrates to humans. In the nematode Caenorhabditis elegans, mutations that eliminate the BK channel, SLO-1, convey dramatic resistance to intoxication by ethanol. We hypothesized that certain conserved amino acids are critical for ethanol modulation, but not for basal channel function. To identify such residues, we screened C. elegans strains with different missense mutations in the SLO-1 channel. A strain with the SLO-1 missense mutation T381I in the RCK1 domain was highly resistant to intoxication. This mutation did not interfere with other BK channel-dependent behaviors, suggesting that the mutant channel retained normal in vivo function. Knock-in of wild-type versions of the worm or human BK channel rescued intoxication and other BK channel-dependent behaviors in a slo-1-null mutant background. In contrast, knock-in of the worm T381I or equivalent human T352I mutant BK channel selectively rescued BK channel-dependent behaviors while conveying resistance to intoxication. Single-channel patch-clamp recordings confirmed that the human BK channel engineered with the T352I missense mutation was insensitive to activation by ethanol, but otherwise had normal conductance, potassium selectivity, and only subtle differences in voltage dependence. Together, our behavioral and electrophysiological results demonstrate that the T352I mutation selectively disrupts ethanol modulation of the BK channel. The T352I mutation may alter a binding site for ethanol and/or interfere with ethanol-induced conformational changes that are critical for behavioral responses to ethanol. PMID:25031399

  12. Functional consequences of site-directed mutation of conserved histidyl residues of the bacterial luciferase alpha subunit.

    PubMed

    Xin, X; Xi, L; Tu, S C

    1991-11-26

    The available sequences for the different bacterial luciferases reveal five conserved histidyl residues at positions 44, 45, 82, 224, and 285 of the alpha subunit. Ten variants of Vibrio harveyi luciferase were obtained by selective site-directed mutations of these five histidines. The essentiality of alpha His44 and alpha His45 was indicated by 4-7 orders of magnitude of bioluminescence activity reductions resulting from the substitution of either histidine by alanine (alpha H44A or alpha H45A), aspartate (alpha H44D or alpha H45D), or lysine (alpha H45K). Moreover, alpha H44A and alpha H45A were distinct from the native luciferase in thermal stabilities. Mutations at the other three positions also resulted in activity reductions ranging from a fewfold to 3 orders of magnitude. Despite these widely different bioluminescence light outputs, mutated luciferases exhibited, in nonturnover in vitro assays, light emission decay rates mostly similar to that of the native luciferase using octanal, decanal, or dodecanal as a substrate. This is attributed to a similarity in the catalytic rate constants of the light-emitting pathway for the native and mutated luciferases, but various mutated luciferases suffer in different degrees from competing dark reaction(s). In accord with this interpretation, the bioluminescence activities of mutated luciferases showed a general parallel with the relative stabilities of their 4a-hydroperoxyflavin intermediate species. Furthermore, the drastically reduced bioluminescence activities for luciferases with the alpha His44 or alpha His45 substituted by aspartate, alanine, or lysine were accompanied by little or no activities for consuming the aldehyde substrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1958663

  13. Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I.

    PubMed

    Colley, William C; van der Merwe, Marie; Vance, John R; Burgin, Alex B; Bjornsti, Mary-Ann

    2004-12-24

    Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding. PMID:15489506

  14. Effect of translational and angular momentum conservation on energy equipartition in microcanonical equilibrium in small clusters.

    PubMed

    Niiyama, Tomoaki; Shimizu, Yasushi; Kobayashi, Taizo R; Okushima, Teruaki; Ikeda, Kensuke S

    2009-05-01

    We investigate numerically and analytically the effects of conservation of total translational and angular momentum on the distribution of kinetic energy among particles in microcanonical particle systems with small number of degrees of freedom, specifically microclusters. Molecular dynamics simulations of microclusters with constant total energy and momenta, using Lennard-Jones, Morse, and Coulomb plus Born-Mayer-type potentials, show that the distribution of kinetic energy among particles can be inhomogeneous and depend on particle mass and position even in thermal equilibrium. Statistical analysis using a microcanonical measure taking into account of the additional conserved quantities gives theoretical expressions for kinetic energy as a function of the mass and position of a particle with only O(1/N;{2}) deviation from the Maxwell-Boltzmann distribution. These expressions fit numerical results well. Finally, we propose an intuitive interpretation for the inhomogeneity of the kinetic energy distributions. PMID:19518410

  15. Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

    SciTech Connect

    Ruel, Nancy . E-mail: n-ruel@northwestern.edu; Zago, Anna . E-mail: anna_zago@acgtinc.com; Spear, Patricia G. . E-mail: p-spear@northwestern.edu

    2006-03-01

    Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity.

  16. Conserved charged residues in the leucine-rich repeat domain of the Ran GTPase activating protein are required for Ran binding and GTPase activation.

    PubMed Central

    Haberland, J; Gerke, V

    1999-01-01

    GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participating in nucleocytoplasmic transport, have been identified in different species ranging from yeast to man. All RanGAPs are characterized by a conserved domain consisting of eight leucine-rich repeats (LRRs) interrupted at two positions by so-called separating regions, the latter being unique for RanGAPs within the family of LRR proteins. The cytosolic RanGAP activity is essential for the Ran GTPase cycle which in turn provides directionality in nucleocytoplasmic transport, but the structural basis for the interaction between Ran and its GAP has not been elucidated. In order to gain a better understanding of this interaction we generated a number of mutant RanGAPs carrying amino acid substitutions in the LRR domain and analysed their complex formation with Ran as well as their ability to stimulate the intrinsic GTPase activity of the G protein. We show that conserved charged residues present in the separating regions of the LRR domain are indispensable for efficient Ran binding and GAP activity. These separating regions contain three conserved arginines which could possibly serve as catalytic residues similar to the arginine fingers identified in GAPs for other small GTPases. However, mutations in two of these arginines do not affect the GAP activity and replacement of the third conserved arginine (Arg91 in human RanGAP) severely interferes not only with GAP activity but also with Ran binding. This indicates that RanGAP-stimulated GTP hydrolysis on Ran does not involve a catalytic arginine residue but requires certain charged residues of the LRR domain of the GAP for mediating the protein-protein interaction. PMID:10527945

  17. The a subunit of the A1AO ATP synthase of Methanosarcina mazei Gö1 contains two conserved arginine residues that are crucial for ATP synthesis.

    PubMed

    Gloger, Carolin; Born, Anna-Katharina; Antosch, Martin; Müller, Volker

    2015-01-01

    Like the evolutionary related F1FO ATP synthases and V1VO ATPases, the A1AO ATP synthases from archaea are multisubunit, membrane-bound transport machines that couple ion flow to the synthesis of ATP. Although the subunit composition is known for at least two species, nothing is known so far with respect to the function of individual subunits or amino acid residues. To pave the road for a functional analysis of A1AO ATP synthases, we have cloned the entire operon from Methanosarcina mazei into an expression vector and produced the enzyme in Escherichia coli. Inverted membrane vesicles of the recombinants catalyzed ATP synthesis driven by NADH oxidation as well as artificial driving forces. [Formula: see text] as well as ΔpH were used as driving forces which is consistent with the inhibition of NADH-driven ATP synthesis by protonophores. Exchange of the conserved glutamate in subunit c led to a complete loss of ATP synthesis, proving that this residue is essential for H+ translocation. Exchange of two conserved arginine residues in subunit a has different effects on ATP synthesis. The role of these residues in ion translocation is discussed. PMID:25724672

  18. Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit.

    PubMed

    García-Sánchez, Sebastián; Sánchez-Cañete, María P; Gamarro, Francisco; Castanys, Santiago

    2014-04-01

    Cdc50 (cell-cycle control protein 50) is a family of conserved eukaryotic proteins that interact with P4-ATPases (phospholipid translocases). Cdc50 association is essential for the endoplasmic reticulum export of P4-ATPases and proper translocase activity. In the present study, we analysed the role of Leishmania infantum LiRos3, the Cdc50 subunit of the P4-ATPase MLF (miltefosine) transporter [LiMT (L. infantum MLF transporter)], on trafficking and complex functionality using site-directed mutagenesis and domain substitution. We identified 22 invariant residues in the Cdc50 proteins from L. infantum, human and yeast. Seven of these residues are found in the extracellular domain of LiRos3, the conservation of which is critical for ensuring that LiMT arrives at the plasma membrane. The substitution of other invariant residues affects complex trafficking to a lesser extent. Furthermore, invariant residues located in the N-terminal cytosolic domain play a role in the transport activity. Partial N-glycosylation of LiRos3 reduces MLF transport and total N-deglycosylation completely inhibits LiMT trafficking to the plasma membrane. One of the N-glycosylation residues is invariant along the Cdc50 family. The transmembrane and exoplasmic domains are not interchangeable with the other two L. infantum Cdc50 proteins to maintain LiMT interaction. Taken together, these findings indicate that both invariant and N-glycosylated residues of LiRos3 are implicated in LiMT trafficking and transport activity. PMID:24447089

  19. Comparative genomics reveals conserved positioning of essential genomic clusters in highly rearranged Thermococcales chromosomes

    PubMed Central

    Cossu, Matteo; Da Cunha, Violette; Toffano-Nioche, Claire; Forterre, Patrick; Oberto, Jacques

    2015-01-01

    The genomes of the 21 completely sequenced Thermococcales display a characteristic high level of rearrangements. As a result, the prediction of their origin and termination of replication on the sole basis of chromosomal DNA composition or skew is inoperative. Using a different approach based on biologically relevant sequences, we were able to determine oriC position in all 21 genomes. The position of dif, the site where chromosome dimers are resolved before DNA segregation could be predicted in 19 genomes. Computation of the core genome uncovered a number of essential gene clusters with a remarkably stable chromosomal position across species, in sharp contrast with the scrambled nature of their genomes. The active chromosomal reorganization of numerous genes acquired by horizontal transfer, mainly from mobile elements, could explain this phenomenon. PMID:26166067

  20. The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

    SciTech Connect

    Liao Maofu; Kielian, Margaret . E-mail: kielian@aecom.yu.edu

    2005-02-05

    The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residues showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion.

  1. Herbicide and cover crop residue integration affects on weed control, quality, and yield in conservation tillage tomato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increased use of conservation tillage in vegetable production requires more information be developed on the role of cover crops in weed control, tomato quality and yield. Three conservation-tillage systems utilizing crimson clover, brassica and cereal rye as winter cover crops were compared to ...

  2. Cotton nitrogen management in a high-residue conservation system: nitrogen source, rate, application method and application timing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conservation tillage systems are promoted in many parts of the world to help achieve environmental sustainability in agriculture. In-service training programs are used to educate extension agents as means to diffuse conservation tillage practices. However, changing extension agents’ attitudes is a c...

  3. Crystallographic and Mutational Studies of Mycobacterium Tuberculosis recA Mini-inteins Suggest a Pivotal Role for a Highly Conserved Aspartate Residue

    SciTech Connect

    Van Roey,P.; Pereira, B.; Li, Z.; Hiraga, K.; Belfort, M.; Derbyshire, V.

    2007-01-01

    The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, {Delta}I-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation ({Delta}I-CM) increased C-terminal cleavage activity. Using the {Delta}I-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, {Delta}{Delta}I{sub hh}-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue {beta}-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of {Delta}I-SM, {Delta}{Delta}I{sub hh}-SM, and two variants, {Delta}{Delta}I{sub hh}-CM and {Delta}{Delta}I{sub hh}, have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions.

  4. Conserved Amino Acid Residues of the NuoD Segment Important for Structure and Function of Escherichia coli NDH-1 (Complex I)

    PubMed Central

    2015-01-01

    The NuoD segment (homologue of mitochondrial 49 kDa subunit) of the proton-translocating NADH:quinone oxidoreductase (complex I/NDH-1) from Escherichia coli is in the hydrophilic domain and bears many highly conserved amino acid residues. The three-dimensional structural model of NDH-1 suggests that the NuoD segment, together with the neighboring subunits, constitutes a putative quinone binding cavity. We used the homologous DNA recombination technique to clarify the role of selected key amino acid residues of the NuoD segment. Among them, residues Tyr273 and His224 were considered candidates for having important interactions with the quinone headgroup. Mutant Y273F retained partial activity but lost sensitivity to capsaicin-40. Mutant H224R scarcely affected the activity, suggesting that this residue may not be essential. His224 is located in a loop near the N-terminus of the NuoD segment (Gly217–Phe227) which is considered to form part of the quinone binding cavity. In contrast to the His224 mutation, mutants G217V, P218A, and G225V almost completely lost the activity. One region of this loop is positioned close to a cytosolic loop of the NuoA subunit in the membrane domain, and together they seem to be important in keeping the quinone binding cavity intact. The structural role of the longest helix in the NuoD segment located behind the quinone binding cavity was also investigated. Possible roles of other highly conserved residues of the NuoD segment are discussed. PMID:25545070

  5. Mutagenic analysis of conserved arginine residues in and around the novel sulfate binding pocket of the human Theta class glutathione transferase T2-2.

    PubMed Central

    Flanagan, J. U.; Rossjohn, J.; Parker, M. W.; Board, P. G.; Chelvanayagam, G.

    1999-01-01

    The human Theta class glutathione transferase GSTT2-2 has a novel sulfatase activity that is not dependent on the presence of a conserved hydrogen bond donor in the active site. Initial homology modeling and the crystallographic studies have identified three conserved Arg residues that contribute to the formation of (Arg107 and Arg239), and entry to (Arg242), a sulfate binding pocket. These residues have been individually mutated to Ala to investigate their potential role in substrate binding and catalysis. The mutation of Arg107 had a significant detrimental effect on the sulfatase reaction, while the Arg242 mutation caused only a small reduction in sulfatase activity. Surprisingly, the Arg239 had an increased activity resulting from a reduction in stability. Thus, Arg239 appears to play a role in maintaining the architecture of the active site. Electrostatic calculations performed on the wild-type and mutant forms of the enzyme are in good agreement with the experimental results. These findings, along with docking studies, suggest that prior to conjugation, the location of 1-menaphthyl sulfate, a model substrate for the sulfatase reaction, is approximately midway between the position ultimately occupied by the naphthalene ring of 1-menaphthylglutathione and the free sulfate. It is further proposed that the Arg residues in and around the sulfate binding pocket have a role in electrostatic substrate recognition. PMID:10548067

  6. NMR WaterLOGSY Reveals Weak Binding of Bisphenol A with Amyloid Fibers of a Conserved 11 Residue Peptide from Androgen Receptor.

    PubMed

    Asencio-Hernández, Julia; Kieffer, Bruno; Delsuc, Marc-André

    2016-01-01

    There is growing evidence that bisphenol A (BPA), a molecule largely released in the environment, has detrimental effects on ecosystems and on human health. It acts as an endocrine disruptor targeting steroid hormone receptors, such as the estrogen receptor (ER), estrogen-related receptor (ERR) and androgen receptor (AR). BPA-derived molecules have recently been shown to interact with the AR N-terminal domain (AR-NTD), which is known to be largely intrinsically disordered. This N-terminal domain contains an 11 residue conserved domain that forms amyloid fibers upon oxidative dimerisation through its strictly conserved Cys240 residue. We investigate here the interaction of BPA, and other potential endocrine disruptors, with AR-NTD amyloid fibers using the WaterLOGSY NMR experiment. We observed a selective binding of these compounds to the amyloid fibers formed by the AR-NTD conserved region and glutamine homopolymers. This observation suggests that the high potency of endocrine disruptors may result, in part, from their ability to bind amyloid forms of nuclear receptors in addition to their cognate binding sites. This property may be exploited to design future therapeutic strategies targeting AR related diseases such as the spinal bulbar muscular atrophy or prostate cancer. The ability of NMR WaterLOGSY experiments to detect weak interactions between small ligands and amyloid fibers may prove to be of particular interest for identifying promising hit molecules. PMID:27583469

  7. Conserved residues Asp16 and Pro24 of TnaC-tRNAPro participate in tryptophan induction of Tna operon expression.

    PubMed

    Cruz-Vera, Luis R; Yanofsky, Charles

    2008-07-01

    In Escherichia coli, interactions between the nascent TnaC-tRNA(Pro) peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNA(Pro) cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased tna operon transcription. Replacing Trp12 of TnaC with any other amino acid residue was previously shown to prevent tryptophan binding and induction of tna operon expression. Genome-wide comparisons of TnaC amino acid sequences identify Asp16 and Pro24, as well as Trp12, as highly conserved TnaC residues. Replacing these residues with other residues was previously shown to influence tryptophan induction of tna operon expression. In this study, in vitro analyses were performed to examine the potential roles of Asp16 and Pro24 in tna operon induction. Replacing Asp16 or Pro24 of TnaC of E. coli with other amino acids established that these residues are essential for free tryptophan binding and inhibition of TnaC-tRNA(Pro) cleavage at the peptidyl transferase center. Asp16 and Pro24 are in fact located in spatial positions corresponding to critical residues of AAP, another ribosome regulatory peptide. Sparsomycin-methylation protection studies further suggested that segments of 23S RNA were arranged differently in ribosomes bearing TnaCs with either the Asp16Ala or the Pro24Ala change. Thus, features of the amino acid sequence of TnaC of the nascent TnaC-tRNA(Pro) peptidyl-tRNA, in addition to the presence of Trp12, are necessary for the nascent peptide to create a tryptophan binding/inhibition site in the translating ribosome. PMID:18424524

  8. Identification of Conserved Amino Acid Residues of the Salmonella σS Chaperone Crl Involved in Crl-σS Interactions ▿

    PubMed Central

    Monteil, Véronique; Kolb, Annie; D'Alayer, Jacques; Beguin, Pierre; Norel, Françoise

    2010-01-01

    Proteins that bind σ factors typically attenuate the function of the σ factor by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary-phase sigma factor σS (RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of σS-dependent genes. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level than rpoS. Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the crl gene from Salmonella enterica serovar Typhimurium and complementation of a Δcrl mutant of Salmonella indicated that substitution of the conserved residues Y22, F53, W56, and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crl-σS interaction and that residues 1 to 71 in σS are dispensable for Crl binding. In Escherichia coli, it has been reported that Crl also interacts with the ferric uptake regulator Fur and that Fur represses crl transcription. However, the Salmonella Crl and Fur proteins did not interact in the BACTH system. In addition, a fur mutation did not have any significant effect on the expression level of Crl in Salmonella. These results suggest that the relationship between Crl and Fur is different in Salmonella and E. coli. PMID:20008066

  9. A conserved proline residue in the leucine zipper region of AtbZIP34 and AtbZIP61 in Arabidopsis thaliana interferes with the formation of homodimer.

    PubMed

    Shen, Huaishun; Cao, Kaiming; Wang, Xiping

    2007-10-19

    Two putative Arabidopsis E group bZIP transcript factors, AtbZIP34 and AtbZIP61, are nuclear-localized and their transcriptional activation domain is in their N-terminal region. By searching GenBank, we found other eight plant homologues of AtbZIP34 and AtbZIP61. All of them have a proline residue in the third heptad of zipper region. Yeast two-hybrid assay and EMSA showed that AtbZIP34 and AtbZIP61 could not form homodimer while their mutant forms, AtbZIP34m and AtbZIP61m, which the proline residue was replaced by an alanine residue in the zipper region, could form homodimer and bind G-box element. These results suggest that the conserved proline residue interferes with the homodimer formation. However, both AtbZIP34 and AtbZIP61 could form heterodimers with members of I group and S group transcription factors in which some members involved in vascular development. So we speculate that AtbZIP34 and AtbZIP61 may participate in plant development via interacting with other group bZIP transcription factors. PMID:17719007

  10. A conserved proline residue in the leucine zipper region of AtbZIP34 and AtbZIP61 in Arabidopsis thaliana interferes with the formation of homodimer

    SciTech Connect

    Shen Huaishun; Cao Kaiming; Wang Xiping

    2007-10-19

    Two putative Arabidopsis E group bZIP transcript factors, AtbZIP34 and AtbZIP61, are nuclear-localized and their transcriptional activation domain is in their N-terminal region. By searching GenBank, we found other eight plant homologues of AtbZIP34 and AtbZIP61. All of them have a proline residue in the third heptad of zipper region. Yeast two-hybrid assay and EMSA showed that AtbZIP34 and AtbZIP61 could not form homodimer while their mutant forms, AtbZIP34m and AtbZIP61m, which the proline residue was replaced by an alanine residue in the zipper region, could form homodimer and bind G-box element. These results suggest that the conserved proline residue interferes with the homodimer formation. However, both AtbZIP34 and AtbZIP61 could form heterodimers with members of I group and S group transcription factors in which some members involved in vascular development. So we speculate that AtbZIP34 and AtbZIP61 may participate in plant development via interacting with other group bZIP transcription factors.

  11. Identification of Conserved Residues in Hepatitis C Virus Envelope Glycoprotein E2 That Modulate Virus Dependence on CD81 and SRB1 Entry Factors

    PubMed Central

    Lavie, Muriel; Sarrazin, Stéphane; Montserret, Roland; Descamps, Véronique; Baumert, Thomas F.; Duverlie, Gilles; Séron, Karin; Penin, François

    2014-01-01

    ABSTRACT In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies. IMPORTANCE Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region

  12. Conserved transmembrane glycine residues in the Shigella flexneri polysaccharide co-polymerase protein WzzB influence protein-protein interactions.

    PubMed

    Papadopoulos, Magdalene; Tran, Elizabeth Ngoc Hoa; Murray, Gerald Laurence; Morona, Renato

    2016-06-01

    The O antigen (Oag) component of lipopolysaccharides (LPS) is crucial for virulence and Oag chain-length regulation is controlled by the polysaccharide co-polymerase class 1 (PCP1) proteins. Crystal structure analyses indicate that structural conservation among PCP1 proteins is highly maintained, however the mechanism of Oag modal-chain-length control remains to be fully elucidated. Shigella flexneri PCP1 protein WzzBSF confers a modal-chain length of 10-17 Oag repeat units (RUs), whereas the Salmonella enterica Typhimurium PCP1 protein WzzBST confers a modal-chain length of ~16-28 Oag RUs. Both proteins share >70 % overall sequence identity and contain two transmembrane (TM1 and TM2) regions, whereby a conserved proline-glycine-rich motif overlapping the TM2 region is identical in both proteins. Conserved glycine residues within TM2 are functionally important, as glycine to alanine substitutions at positions 305 and 311 confer very short Oag modal-chain length (~2-6 Oag RUs). In this study, WzzBSF was co-expressed with WzzBST in S. flexneri and a single intermediate modal-chain length of ~11-21 Oag RUs was observed, suggesting the presence of Wzz:Wzz interactions. Interestingly, co-expression of WzzBSF with WzzBG305A/G311A conferred a bimodal LPS Oag chain length (despite over 99 % protein sequence identity), and we hypothesized that the proteins fail to interact. Co-purification assays detected His6-WzzBSF co-purifying with FLAG-tagged WzzBST but not with FLAG-tagged WzzBG305A/G311A, supporting our hypothesis. These data indicate that the conserved glycine residues in TM2 are involved in Wzz:Wzz interactions, and provide insight into key interactions that drive Oag modal length control. PMID:27028755

  13. Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m6A methyltransferase ErmC'.

    PubMed

    Maravić, Gordana; Feder, Marcin; Pongor, Sándor; Flögel, Mirna; Bujnicki, Janusz M

    2003-09-01

    Methyltransferases (MTases) from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B antibiotics. Despite the available structural data and functional analyses on the level of the RNA substrate, still very little is known about the mechanism of rRNA:adenine-N(6) methylation. Only predictions regarding various aspects of this reaction have been made based on the analysis of the crystal structures of methyltransferase ErmC' (without the RNA) and their comparison with the crystallographic and biochemical data for better studied DNA:m(6)A MTases. To validate the structure-based predictions of presumably essential residues in the catalytic pocket of ErmC', we carried out the site-directed mutagenesis and studied the function of the mutants in vitro and in vivo. Our results indicate that the active site of rRNA:m(6)A MTases is much more tolerant to amino acid substitutions than the active site of DNA:m(6)A MTases. Only the Y104 residue implicated in stabilization of the target base was found to be indispensable. Remarkably, the N101 residue from the "catalytic" motif IV and two conserved residues that form the floor (F163) and one of the walls (N11) of the base-binding site are not essential for catalysis in ErmC'. This somewhat surprising result is discussed in the light of the available structural data and in the phylogenetic context of the Erm family. PMID:12946350

  14. Conserved Cysteine Residue in the DNA-Binding Domain of the Bovine Papillomavirus Type 1 E2 Protein Confers Redox Regulation of the DNA- Binding Activity in Vitro

    NASA Astrophysics Data System (ADS)

    McBride, Alison A.; Klausner, Richard D.; Howley, Peter M.

    1992-08-01

    The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfydryl group(s) may be protected by DNA binding. Sensitivity to sulfydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

  15. Structure-function relationship in the globular type III antifreeze protein: identification of a cluster of surface residues required for binding to ice.

    PubMed Central

    Chao, H.; Sönnichsen, F. D.; DeLuca, C. I.; Sykes, B. D.; Davies, P. L.

    1994-01-01

    Antifreeze proteins (AFPs) depress the freezing point of aqueous solutions by binding to and inhibiting the growth of ice. Whereas the ice-binding surface of some fish AFPs is suggested by their linear, repetitive, hydrogen bonding motifs, the 66-amino-acid-long Type III AFP has a compact, globular fold without any obvious periodicity. In the structure, 9 beta-strands are paired to form 2 triple-stranded antiparallel sheets and 1 double-stranded antiparallel sheet, with the 2 triple sheets arranged as an orthogonal beta-sandwich (Sönnichsen FD, Sykes BD, Chao H, Davies PL, 1993, Science 259:1154-1157). Based on its structure and an alignment of Type III AFP isoform sequences, a cluster of conserved, polar, surface-accessible amino acids (N14, T18, Q44, and N46) was noted on and around the triple-stranded sheet near the C-terminus. At 3 of these sites, mutations that switched amide and hydroxyl groups caused a large decrease in antifreeze activity, but amide to carboxylic acid changes produced AFPs that were fully active at pH 3 and pH 6. This is consistent with the observation that Type III AFP is optimally active from pH 2 to pH 11. At a concentration of 1 mg/mL, Q44T, N14S, and T18N had 50%, 25%, and 10% of the activity of wild-type antifreeze, respectively. The effects of the mutations were cumulative, such that the double mutant N14S/Q44T had 10% of the wild-type activity and the triple mutant N14S/T18N/Q44T had no activity. All mutants with reduced activity were shown to be correctly folded by NMR spectroscopy. Moreover, a complete characterization of the triple mutant by 2-dimensional NMR spectroscopy indicated that the individual and combined mutations did not significantly alter the structure of these proteins. These results suggest that the C-terminal beta-sheet of Type III AFP is primarily responsible for antifreeze activity, and they identify N14, T18, and Q44 as key residues for the AFP-ice interaction. PMID:7849594

  16. Roles of a conserved arginine residue of DsbB in linking protein disulfide-bond-formation pathway to the respiratory chain of Escherichia coli

    PubMed Central

    Kadokura, Hiroshi; Bader, Martin; Tian, Hongping; Bardwell, James C. A.; Beckwith, Jon

    2000-01-01

    The active-site cysteines of DsbA, the periplasmic disulfide-bond-forming enzyme of Escherichia coli, are kept oxidized by the cytoplasmic membrane protein DsbB. DsbB, in turn, is oxidized by two kinds of quinones (ubiquinone for aerobic and menaquinone for anaerobic growth) in the electron-transport chain. We describe the isolation of dsbB missense mutations that change a highly conserved arginine residue at position 48 to histidine or cysteine. In these mutants, DsbB functions reasonably well aerobically but poorly anaerobically. Consistent with this conditional phenotype, purified R48H exhibits very low activity with menaquinone and an apparent Michaelis constant (Km) for ubiquinone seven times greater than that of the wild-type DsbB, while keeping an apparent Km for DsbA similar to that of wild-type enzyme. From these results, we propose that this highly conserved arginine residue of DsbB plays an important role in the catalysis of disulfide bond formation through its role in the interaction of DsbB with quinones. PMID:11005861

  17. Conserved Ser residues in the basic region of the bZIP-type transcription factor HBP-1a(17): importance in DNA binding and possible targets for phosphorylation.

    PubMed

    Meshi, T; Moda, I; Minami, M; Okanami, M; Iwabuchi, M

    1998-01-01

    HBP-1a(17) is representative of a group of plant bZIP-type transcription factors which includes HBP-1a proteins and G-box-binding factors. We found kinase activity in wheat nuclear extract that phosphorylated HBP-1a(17). Experiments using recombinant HBP-1a(17) derivatives as substrates revealed that all three of the Ser residues in the basic region, Ser-261, Ser-265, and Ser-269, were phosphorylated in a Ca(2+)-stimulated manner. DNA-binding analysis of mutants with a Ser-to-Glu change, prepared to mimic the phosphorylated proteins, indicated that introduction of a negative charge at position 265 or 269 prevents HBP-1a(17) from binding DNA not only in the homodimer of mutants but also in heterodimers with a wild-type protein. It is therefore suggested that the phosphorylation regulates the function of HBP-1a(17) at least at the level of DNA binding. Since Ser-265 and Ser-269 are highly conserved among the plant bZIP-type factors known to date, a common Ca(2+)-mediated regulatory mechanism may exert an effect on the bZIP-type factors through phosphorylation of these conserved Ser residues. PMID:9484468

  18. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production.

    PubMed

    Tay, Moon Y F; Smith, Kate; Ng, Ivan H W; Chan, Kitti W K; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Luo, Dahai; Jans, David A; Forwood, Jade K; Vasudevan, Subhash G

    2016-09-01

    Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization. PMID:27622521

  19. Leucine-684: A conserved residue of an AMP-acetyl CoA synthetase (AceCS) from Leishmania donovani is involved in substrate recognition, catalysis and acetylation.

    PubMed

    Soumya, Neelagiri; Tandan, Hitendra; Damre, Mangesh V; Gangwal, Rahul P; Sangamwar, Abhay T; Singh, Sushma

    2016-04-15

    AMP-acetyl CoA synthetase (AMP-AceCS) is a key enzyme which catalyzes the activation of acetate to acetyl CoA, an important intermediate at the cross roads of various anabolic and catabolic pathways. Multiple sequence alignment of Leishmania donovani AceCS with other organisms revealed the presence of a highly conserved leucine residue at 684 position which is known to be crucial for acetylation by protein acetyl transferases in other organisms. In an attempt to understand the role of leucine residue at 684 position in L. donovani acetyl CoA synthetase (LdAceCS), it was mutated to proline (P) by site directed mutagenesis. Kinetic analysis of the L684P-LdAceCS mutant revealed approximately two fold increased binding affinity with acetate, whereas fivefold decreased affinity was observed with ATP. There was insignificant change in secondary structure as revealed by CD however, two fold decreased fluorescence intensity was observed at an emission maxima of 340nm. Interestingly, L684P mutation abolished the acetylation of the mutant enzyme indicating the importance of L684 in acetylation of the enzyme. Changes in biochemical parameters of the mutant protein were validated by homology modeling of the wild type and mutant LdAceCS enzyme using Salmonella enterica AceCS crystal structure as template. Our data provides evidence for the role of leucine 684 residue in substrate recognition, catalysis and acetylation of the AceCS enzyme. PMID:26794803

  20. A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

    SciTech Connect

    Wu, Di Wu, Shian

    2013-04-19

    Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

  1. Conserved residues flanking the thiol/disulfide centers of protein disulfide isomerase are not essential for catalysis of thiol/disulfide exchange.

    PubMed

    Lu, X; Gilbert, H F; Harper, J W

    1992-05-01

    Protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds by increasing the rate of disulfide bond rearrangements which normally occur during the folding process. The amino acid sequences of the N- and C-terminal redox active sites (PWCGHCK) in PDI are completely conserved from yeast to man and display considerable identity with the redox-active center of thioredoxin (EWCGPCK). Available data indicate that the two thiol/disulfide centers of PDI can function independently in the isomerase reaction and that the cysteine residues in each active site are essential for catalysis. To evaluate the role of residues flanking the active-site cysteines of PDI in function, a variety of mutations were introduced into the N-terminal active site of PDI within the context of both a functional C-terminal active site and an inactive C-terminal active site in which serine residues replaced C379 and C382. Replacement of non-cysteine residues (W34 to Ser, G36 to Ala, and K39 to Arg) resulted in only a modest reduction in catalytic activity in both the oxidative refolding of RNase A and the reduction of insulin (10-27%), independent of the status of the C-terminal active site. A somewhat larger effect was observed with the H37P mutation where approximately 80% of the activity attributable to the N-terminal domain (approximately 40%) was lost. However, the H37P mutant N-terminal site expressed within the context of an inactive C-terminal domain exhibits 30% activity, approximately 70% of the activity of the N-terminal site alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1567868

  2. The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion

    PubMed Central

    Etienne, Loïc; Blanchard, Emmanuelle; Boyer, Audrey; Desvignes, Virginie; Gaillard, Julien; Meunier, Jean-Christophe; Roingeard, Philippe; Hourioux, Christophe

    2015-01-01

    Hepatitis C virus (HCV) assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD) surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER) membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis. PMID:26339783

  3. Residue Substitutions Near the Redox Center of Bacillus subtilis Spx Affect RNA Polymerase Interaction, Redox Control, and Spx-DNA Contact at a Conserved cis-Acting Element

    PubMed Central

    Lin, Ann A.; Walthers, Don

    2013-01-01

    Spx, a member of the ArsC protein family, is a regulatory factor that interacts with RNA polymerase (RNAP). It is highly conserved in Gram-positive bacteria and controls transcription on a genome-wide scale in response to oxidative stress. The structural requirements for RNAP interaction and promoter DNA recognition by Spx were examined through mutational analysis. Residues near the CxxC redox disulfide center of Spx functioned in RNAP α subunit interaction and in promoter DNA binding. R60E and C10A mutants were shown previously to confer defects in transcriptional activation, but both were able to interact with RNAP. R92, which is conserved in ArsC-family proteins, is likely involved in redox control of Spx, as the C10A mutation, which blocks disulfide formation, was epistatic to the R92A mutation. The R91A mutation reduced transcriptional activation and repression, suggesting a defect in RNAP interaction, which was confirmed by interaction assays using an epitope-tagged mutant protein. Protein-DNA cross-linking detected contact between RNAP-bound Spx and the AGCA element at −44 that is conserved in Spx-controlled genes. This interaction caused repositioning of the RNAP σA subunit from a −35-like element upstream of the trxB (thioredoxin reductase) promoter to positions −36 and −11 of the core promoter. The study shows that RNAP-bound Spx contacts a conserved upstream promoter sequence element when bound to RNAP. PMID:23813734

  4. The biliverdin chromophore binds covalently to a conserved cysteine residue in the N-terminus of Agrobacterium phytochrome Agp1.

    PubMed

    Lamparter, Tilman; Carrascal, Montserrat; Michael, Norbert; Martinez, Enriqueta; Rottwinkel, Gregor; Abian, Joaquin

    2004-03-30

    Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction. PMID:15035636

  5. DHARMA - Discriminant hyperplane abstracting residuals minimization algorithm for separating clusters with fuzzy boundaries. [data points pattern recognition technique

    NASA Technical Reports Server (NTRS)

    Dasarathy, B. V.

    1976-01-01

    Learning of discriminant hyperplanes in imperfectly supervised or unsupervised training sample sets with unreliably labeled samples along the fuzzy joint boundaries between sample clusters is discussed, with the discriminant hyperplane designed to be a least-squares fit to the unreliably labeled data points. (Samples along the fuzzy boundary jump back and forth from one cluster to the other in recursive cluster stabilization and are considered unreliably labeled.) Minimization of the distances of these unreliably labeled samples from the hyperplanes does not sacrifice the ability to discriminate between classes represented by reliably labeled subsets of samples. An equivalent unconstrained linear inequality problem is formulated and algorithms for its solution are indicated. Landsat earth sensing data were used in confirming the validity and computational feasibility of the approach, which should be useful in deriving discriminant hyperplanes separating clusters with fuzzy boundaries, given supervised training sample sets with unreliably labeled boundary samples.

  6. A light-dependent complementation system for analysis of NADPH:protochlorophyllide oxidoreductase: Identification and mutagenesis of two conserved residues that are essential for enzyme activity

    SciTech Connect

    Wilks, H.M.; Timko, M.P.

    1995-01-31

    Protochlorophyllide reductase (NADPH:protochlorophyllide oxidoreductase; EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory step in the chlorophyll biosynthetic pathway. We have developed an expression system in which the protochlorophyllide reductase from pea (Pisum sativum L.) is used to complement protochlorophyllide reduction mutants in the photosynthetic bacterium Rhodobacter capsulatus, allowing analysis of wild-type and mutant forms of the enzyme. By protein sequence comparisons, we have identified the plant protochlorophyllide reductases as belonging to the family of short-chain alcohol dehydrogenases. Based on our protein sequence alignments, we have identified and mutated two conserved residues (Tyr-275 and Lys-279) within the proposed active site of the enzyme and shown that they are critical for activity. A model of the enzyme reaction mechanism for light-dependent protochlorophyllide reduction is proposed. 33 refs., 5 figs.

  7. Extended, Dusty Star Formation Fueled by a Residual Cooling Flow in the Cluster of Galaxies Sérsic 159-03

    NASA Astrophysics Data System (ADS)

    McDonald, M.; Werner, N.; Oonk, J. B. R.; Veilleux, S.

    2015-05-01

    While the cooling of the hot intracluster medium (ICM) in the cores of galaxy clusters is mostly counteracted by heating from the central active galactic nucleus (AGN), the balance is not perfect. This can lead to residual cooling flows and low-level star formation, the physics of which is not well understood. Here we present a detailed study of the residual cooling flow in the center of the low mass galaxy cluster Sérsic 159-03 (A S1101; z = 0.058) using far-ultraviolet imaging from the Hubble Space Telescope and far-IR (FIR) spectroscopy and photometry from the Herschel Space Observatory, along with a wealth of archival data. We detect extended emission at UV, FIR, and [C ii], indicating a star formation rate of ˜1-3 {{M}⊙ } yr-1, depending on the indicator and assumptions made. The most recently formed stars (as indicated by high Hα/UV ratios) appear spatially coincident with the lowest entropy ICM. We speculate that this low-entropy gas has been displaced by the central AGN ˜7.5 kpc north of the cD galaxy. These data suggest that the displacement of the cooling core from the direct vicinity of the central AGN can temporarily break the feedback cycle and lead to cooling and star formation that is offset from the center of the galaxy. We find an abundance (˜107 {{M}⊙ }) of cold (20 K) dust in the center of the cluster and a second FIR peak ˜30 kpc to the north of the central galaxy. If confirmed to be associated with the cooling filaments, this would be the most extended complex of dust yet found in a cool core cluster.

  8. A cluster of charged and aromatic residues in the C-terminal portion of maltoporin participates in sugar binding and uptake.

    PubMed

    Charbit, A; Wang, J; Michel, V; Hofnung, M

    1998-11-01

    The maltoporin LamB of Escherichia coli K12 is a trimeric protein which facilitates the diffusion of maltose and maltodextrins through the bacterial outer membrane, and also acts as a non-specific porin for small hydrophilic molecules as well as a receptor for phages. Loop L9 (residues 375 to 405) is the most distal and largest surface-exposed loop of LamB. It comprises a central portion, which varies in size and sequence in the maltoporins of known sequence, flanked by two conserved regions containing charged and aromatic residues. In order to identify the residues within the proximal region that are specifically involved in sugar utilization, we used site-directed mutagenesis to change, individually, each of the charged (five) and aromatic (three) residues in the region 371 to 379 into alanine. None of the eight single amino acid substitutions affected the phage receptor activity of LamB. In contrast, they all affected, to variable extents, maltoporin functions. For all the mutants, very good correlations were observed between the effects on sugar binding and on in vivo uptake. In no case were maltoporin functions completely abolished. Mutants E374 A and W376 A were the most impaired (with over 60% reduction in dextrin binding and in vivo uptake of maltose and maltopentaose). These two mutations also led to an increased bacterial sensitivity to bacitracin and vancomycin. The functional and structural implications are discussed. PMID:9862470

  9. AtNHX5 and AtNHX6 Control Cellular K+ and pH Homeostasis in Arabidopsis: Three Conserved Acidic Residues Are Essential for K+ Transport

    PubMed Central

    Wang, Liguang; Wu, Xuexia; Liu, Yafen; Qiu, Quan-Sheng

    2015-01-01

    AtNHX5 and AtNHX6, the endosomal Na+,K+/H+ antiporters in Arabidopsis, play an important role in plant growth and development. However, their function in K+ and pH homeostasis remains unclear. In this report, we characterized the function of AtNHX5 and AtNHX6 in K+ and H+ homeostasis in Arabidopsis. Using a yeast expression system, we found that AtNHX5 and AtNHX6 recovered tolerance to high K+ or salt. We further found that AtNHX5 and AtNHX6 functioned at high K+ at acidic pH while AtCHXs at low K+ under alkaline conditions. In addition, we showed that the nhx5 nhx6 double mutant contained less K+ and was sensitive to low K+ treatment. Overexpression of AtNHX5 or AtNHX6 gene in nhx5 nhx6 recovered root growth to the wild-type level. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for K+ homeostasis and plant growth. nhx5 nhx6 had a reduced vacuolar and cellular pH as measured with the fluorescent pH indicator BCECF or semimicroelectrode. We further show that AtNHX5 and AtNHX6 are localized to Golgi and TGN. Taken together, AtNHX5 and AtNHX6 play an important role in K+ and pH homeostasis in Arabidopsis. Three conserved acidic residues are essential for K+ transport. PMID:26650539

  10. Residues in three conserved regions of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase are required for quaternary structure

    SciTech Connect

    Fitchen, J.H.; McIntosh, L. ); Knight, S.; Andersson, I.; Branden, C.I. )

    1990-08-01

    To explore the role of individual residues in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, small subunits with single amino acid substitutions in three regions of relative sequence conservation were produced by directed mutagenesis of the rbcS gene from Anabaena 7120. These altered small subunits were cosythesized with large subunits (from an expressed Anabaena rbcL gene) in Escherichia coli. Mutants were analyzed for effects on quaternary structure and catalytic activity. Changing Glu-13S (numbering used is that of the spinach enzyme) to Val, Trp-67S to Arg, Pro-73S to His, or Tyr-98S to Asn prevented accumulation of stable holoenzyme. Interpretation of these results using a model for the three-dimensional structure of the spinach enzyme based on x-ray crystallographic data suggests that our small subunit mutants containing substitutions at positions 13S and 67S probably do not assemble because of mispairing or nonpairing of charged residues on the interfacing surfaces of the large and small subunits. The failure of small subunits substituted at positions 73S or 98S to assemble correctly may result from disruption of intersubunit or intrasubunit hydrophobic pockets, respectively.

  11. The conserved Trp114 residue of thioredoxin reductase 1 has a redox sensor-like function triggering oligomerization and crosslinking upon oxidative stress related to cell death

    PubMed Central

    Xu, J; Eriksson, S E; Cebula, M; Sandalova, T; Hedström, E; Pader, I; Cheng, Q; Myers, C R; Antholine, W E; Nagy, P; Hellman, U; Selivanova, G; Lindqvist, Y; Arnér, E S J

    2015-01-01

    The selenoprotein thioredoxin reductase 1 (TrxR1) has several key roles in cellular redox systems and reductive pathways. Here we discovered that an evolutionarily conserved and surface-exposed tryptophan residue of the enzyme (Trp114) is excessively reactive to oxidation and exerts regulatory functions. The results indicate that it serves as an electron relay communicating with the FAD moiety of the enzyme, and, when oxidized, it facilitates oligomerization of TrxR1 into tetramers and higher multimers of dimers. A covalent link can also be formed between two oxidized Trp114 residues of two subunits from two separate TrxR1 dimers, as found both in cell extracts and in a crystal structure of tetrameric TrxR1. Formation of covalently linked TrxR1 subunits became exaggerated in cells on treatment with the pro-oxidant p53-reactivating anticancer compound RITA, in direct correlation with triggering of a cell death that could be prevented by antioxidant treatment. These results collectively suggest that Trp114 of TrxR1 serves a function reminiscent of an irreversible sensor for excessive oxidation, thereby presenting a previously unrecognized level of regulation of TrxR1 function in relation to cellular redox state and cell death induction. PMID:25611390

  12. Redundancy of the conserved His residue in Azotobacter vinelandii NifL, a histidine autokinase homologue which regulates transcription of nitrogen fixation genes.

    PubMed

    Woodley, P; Drummond, M

    1994-08-01

    The NifL protein of Azotobacter vinelandii inhibits NifA, the activator of nif (nitrogen fixation) transcription, in response to oxygen and fixed nitrogen. NifL shows strong homology in its C-terminal domain to the histidine autokinase domains of the canonical two-component sensor proteins, including the region around His-304, which corresponds to the residue known to be phosphorylated in other systems. To examine the mechanism of sensory transduction by NifL, mutations encoding 10 substitutions for His-304 were introduced into the A. vinelandii chromosome. Regulation of nif transcription was measured using acetylene reduction and RNA blots. The substitutions His-304-->Arg and His-304-->Pro impaired regulation by both fixed nitrogen and oxygen, but substitution of Ala, Phe, Ile, Lys, Asn, Ser, Thr, Val had no effect. None of the mutants, including His-304-->Arg and His-304-->Pro, excreted ammonium during diazotrophy, a phenotype of nifL deletion mutants, suggesting that the molecular basis of this effect differs from that responsible for the inhibition of nif transcription. The data show conclusively that phosphorylation of His-304 is not essential for any of the known functions of A. vinelandii NifL. Homology to the family of histidine autokinases is therefore inadequate evidence for a mechanism of sensory transduction involving phosphorylation of the conserved histidine residue. PMID:7997174

  13. Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development

    PubMed Central

    Liu, Chia-Lin; Hung, Hui-Chen; Lo, Shou-Chen; Chiang, Ching-Hui; Chen, I-Jung; Hsu, John T.-A.; Hou, Ming-Hon

    2016-01-01

    Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus–infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP’s RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus. PMID:26916998

  14. Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3*

    PubMed Central

    Slugoski, Melissa D.; Smith, Kyla M.; Ng, Amy M. L.; Yao, Sylvia Y. M.; Karpinski, Edward; Cass, Carol E.; Baldwin, Stephen A.; Young, James D.

    2009-01-01

    Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na+ and H+ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na+/nucleoside and H+/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H+ dependence (all mutants), shift in Na+:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na+-specific hCNT1, uncoupled Na+ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine. PMID:19380587

  15. Inactivation of influenza virus haemagglutinin by chlorine dioxide: oxidation of the conserved tryptophan 153 residue in the receptor-binding site.

    PubMed

    Ogata, Norio

    2012-12-01

    Airborne influenza virus infection of mice can be prevented by gaseous chlorine dioxide (ClO(2)). This study demonstrated that ClO(2) abolished the function of the haemagglutinin (HA) of influenza A virus (H1N1) in a concentration-, time- and temperature-dependent manner. The IC(50) during a 2 min reaction with ClO(2) at 25 °C was 13.7 µM, and the half-life time of HA with 100 µM ClO(2) at 25 °C was 19.5 s. Peptides generated from a tryptic digest of ClO(2)-treated virus were analysed by mass spectrometry. An HA fragment, (150)NLLWLTGK(157) was identified in which the tryptophan residue (W153) was 32 mass units greater than expected. The W153 residue of this peptide, which is derived from the central region of the receptor-binding site of HA, is highly conserved. It was shown that W153 was oxidized to N-formylkynurenine in ClO(2)-treated virus. It was concluded that the inactivation of influenza virus by ClO(2) is caused by oxidation of W153 in HA, thereby abolishing its receptor-binding ability. PMID:22933663

  16. Insights into the smooth-to-rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members.

    PubMed

    Bernut, Audrey; Viljoen, Albertus; Dupont, Christian; Sapriel, Guillaume; Blaise, Mickaël; Bouchier, Christiane; Brosch, Roland; de Chastellier, Chantal; Herrmann, Jean-Louis; Kremer, Laurent

    2016-03-01

    In mycobacteria, MmpL proteins represent key components that participate in the biosynthesis of the complex cell envelope. Whole genome analysis of a spontaneous rough morphotype variant of Mycobacterium abscessus subsp. bolletii identified a conserved tyrosine that is crucial for the function of MmpL family proteins. Isogenic smooth (S) and rough (R) variants differed by a single mutation linked to a Y842H substitution in MmpL4a. This mutation caused a deficiency in glycopeptidolipid production/transport in the R variant and a gain in the capacity to produce cords in vitro. In zebrafish, increased virulence of the M. bolletii R variant over the parental S strain was found, involving massive production of serpentine cords, abscess formation and rapid larval death. Importantly, this finding allowed us to demonstrate an essential role of Tyr842 in several different MmpL proteins, including Mycobacterium tuberculosis MmpL3. Structural homology models of MmpL4a and MmpL3 identified two additional critical residues located in the transmembrane regions TM10 and TM4 that are facing each other. We propose that these central residues are part of the proton-motive force that supplies the energy for substrate transport. Hence, we provide important insights into mechanistic/structural aspects of MmpL proteins as lipid transporters and virulence determinants in mycobacteria. PMID:26585558

  17. Systematic Analysis of the Amino Acid Residues of Human Papillomavirus Type 16 E7 Conserved Region 3 Involved in Dimerization and Transformation ▿

    PubMed Central

    Todorovic, Biljana; Massimi, Paola; Hung, Katherine; Shaw, Gary S.; Banks, Lawrence; Mymryk, Joe S.

    2011-01-01

    The human papillomavirus (HPV) E7 oncoprotein exists as a dimer and acts by binding to many cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. Dimerization of E7 is attributed primarily to the C-terminal domain, referred to as conserved region 3 (CR3). CR3 is highly structured and is necessary for E7's transformation ability. It is also required for binding of numerous E7 cellular targets. To systematically analyze the molecular mechanisms by which HPV16 E7 CR3 contributes to carcinogenesis, we created a comprehensive panel of mutations in residues predicted to be exposed on the surface of CR3. We analyzed our novel collection of mutants, as well as mutants targeting predicted hydrophobic core residues of the dimer, for the ability to dimerize. The same set of mutants was also assessed functionally for transformation capability in a baby rat kidney cell assay in conjugation with activated ras. We show that some mutants of HPV16 E7 CR3 failed to dimerize yet were still able to transform baby rat kidney cells. Our results identify several novel E7 mutants that abrogate transformation and also indicate that E7 does not need to exist as a stable dimer in order to transform cells. PMID:21775462

  18. Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development.

    PubMed

    Liu, Chia-Lin; Hung, Hui-Chen; Lo, Shou-Chen; Chiang, Ching-Hui; Chen, I-Jung; Hsu, John T-A; Hou, Ming-Hon

    2016-01-01

    Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus-infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP's RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus. PMID:26916998

  19. The Interdomain Linker of AAV-2 Rep68 Is an Integral Part of Its Oligomerization Domain: Role of a Conserved SF3 Helicase Residue in Oligomerization

    PubMed Central

    Zarate-Perez, Francisco; Bardelli, Martino; Burgner, John W.; Villamil-Jarauta, Maria; Das, Kanni; Kekilli, Demet; Mansilla-Soto, Jorge; Linden, R. Michael; Escalante, Carlos R.

    2012-01-01

    The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains. PMID:22719256

  20. Comparative losses of glyphosate and selected residual herbicides in surface runoff from conservation-tilled watersheds planted with corn or soybean.

    PubMed

    Shipitalo, Martin J; Owens, Lloyd B

    2011-01-01

    Residual herbicides regularly used in conjunction with conservation tillage to produce corn ( L.) and soybean [ (L.) Merr] are often detected in surface water at concentrations that exceed their U.S. maximum contaminant levels (MCL) and ecological standards. These risks might be reduced by planting glyphosate-tolerant varieties of these crops and totally or partially replacing the residual herbicides alachlor, atrazine, linuron, and metribuzin with glyphosate, a contact herbicide that has a short half-life and is strongly sorbed to soil. Therefore, we applied both herbicide types at typical rates and times to two chisel-plowed and two no-till watersheds in a 2-yr corn/soybean rotation and at half rates to three disked watersheds in a 3-yr corn/soybean/wheat-red clover ( L.- L.) rotation and monitored herbicide losses in surface runoff for three crop years. Average dissolved glyphosate loss for all tillage practices, as a percentage of the amount applied, was significantly less ( ≤ 0.05) than the losses of atrazine (21.4x), alachlor (3.5x), and linuron (8.7x) in corn-crop years. Annual, flow-weighted, concentration of atrazine was as high as 41.3 μg L, much greater than its 3 μg L MCL. Likewise, annual, flow-weighted alachlor concentration (MCL = 2 μg L) was as high as 11.2 and 4.9 μg L in corn- and soybean-crop years, respectively. In only one runoff event during the 18 watershed-years it was applied did glyphosate concentration exceed its 700 μg L MCL and the highest, annual, flow-weighted concentration was 3.9 μg L. Planting glyphosate-tolerant corn and soybean and using glyphosate in lieu of some residual herbicides should reduce the impact of the production of these crops on surface water quality. PMID:21712598

  1. Non-Conserved Residues in Clostridium acetobutylicum tRNAAla Contribute to tRNA Tuning for Efficient Antitermination of the alaS T Box Riboswitch

    PubMed Central

    Liu, Liang-Chun; Grundy, Frank J.; Henkin, Tina M.

    2015-01-01

    The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient antitermination. PMID:26426057

  2. Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading.

    PubMed

    Felczak, Magdalena M; Sage, Jay M; Hupert-Kocurek, Katarzyna; Aykul, Senem; Kaguni, Jon M

    2016-02-26

    The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature. PMID:26728455

  3. Site-specific mutations of conserved residues in the phosphate-binding loop of the Arabidopsis UMP/CMP kinase alter ATP and UMP binding.

    PubMed

    Zhou, L; Thornburg, R

    1998-10-15

    All eukaryotic UMP/CMP kinases contain a glycine-rich sequence GGPG(S/A)GK at the N-terminus. This sequence is homologous to the conserved sequence GXXGXGK found in other ATP-binding proteins. To study the role of this conserved sequence in Arabidopsis UMP/CMP kinase, five conserved residues were mutated by site-directed mutagenesis to generate seven mutant enzymes: G21A, G22A, G24A, G26A, K27R, K27M, and K27E. The G21A and G26A mutants were degraded during the purification phase and were thus unable to be purified. Kinetic studies on the other mutants, when compared to studies on the wild-type enzyme, revealed that this sequence is important for ATP binding and enzyme catalysis. All mutants had a decreased kcat/KATPm value. The G22A and G24A mutants had about half of the kcat value of wildtype and 3.9-fold and 3.3-fold increases in KATPm values, respectively. The kcat/KATPm values in the K27M and K27E mutants were changed significantly and decreased by 1000-fold and 2600-fold, respectively. The removal of the terminal positive charge of Lys27 in the K27M and K27E mutants resulted in 20% of the kcat value of wildtype. However, both mutants had a remarkable increase in KATPm value by 241-fold and 552-fold, respectively. Therefore, the positive charge of Lys27 plays an important role on both ATP binding and enzyme catalysis. Interestingly, the results also showed that the mutations that affected ATP binding also had an effect on UMP binding. PMID:9784243

  4. G glycoprotein amino acid residues required for human monoclonal antibody RAB1 neutralization are conserved in rabies virus street isolates.

    PubMed

    Wang, Yang; Rowley, Kirk J; Booth, Brian J; Sloan, Susan E; Ambrosino, Donna M; Babcock, Gregory J

    2011-08-01

    Replacement of polyclonal anti-rabies immunoglobulin (RIG) used in rabies post-exposure prophylaxis (PEP) with a monoclonal antibody will eliminate cost and availability constraints that currently exist using RIG in the developing world. The human monoclonal antibody RAB1 has been shown to neutralize all rabies street isolates tested; however for the laboratory-adapted fixed strain, CVS-11, mutation in the G glycoprotein of amino acid 336 from asparagine (N) to aspartic acid (D) resulted in resistance to neutralization. Interestingly, this same mutation in the G glycoprotein of a second laboratory-adapted fixed strain (ERA) did not confer resistance to RAB1 neutralization. Using cell surface staining and lentivirus pseudotyped with rabies virus G glycoprotein (RABVpp), we identified an amino acid alteration in CVS-11 (K346), not present in ERA (R346), which was required in combination with D336 to confer resistance to RAB1. A complete analysis of G glycoprotein sequences from GenBank demonstrated that no identified rabies isolates contain the necessary combination of G glycoprotein mutations for resistance to RAB1 neutralization, consistent with the broad neutralization of RAB1 observed in direct viral neutralization experiments with street isolates. All combinations of amino acids 336 and 346 reported in the sequence database were engineered into the ERA G glycoprotein and RAB1 was able to neutralize RABVpp bearing ERA G glycoprotein containing all known combinations at these critical residues. These data demonstrate that RAB1 has the capacity to neutralize all identified rabies isolates and a minimum of two distinct mutations in the G glycoprotein are required for abrogation of RAB1 neutralization. PMID:21693135

  5. Aqueous and tissue residue-based interspecies correlation estimation models provide conservative hazard estimates for aromatic compounds.

    PubMed

    Bejarano, Adriana C; Barron, Mace G

    2016-01-01

    Interspecies correlation estimation (ICE) models were developed for 30 nonpolar aromatic compounds to allow comparison of prediction accuracy between 2 data compilation approaches. Type 1 models used data combined across studies, and type 2 models used data combined only within studies. Target lipid (TLM) ICE models were also developed using target lipid concentrations of the type 2 model dataset (type 2-TLM). Analyses were performed to assess model prediction uncertainty introduced by each approach. Most statistically significant models (90%; 266 models total) had mean square errors < 0.27 and adjusted coefficients of determination (adj R(2) ) > 0.59, with the lowest amount of variation in mean square errors noted for type 2-TLM followed by type 2 models. Cross-validation success (>0.62) across most models (86% of all models) confirmed the agreement between ICE predicted and observed values. Despite differences in model predictive ability, most predicted values across all 3 ICE model types were within a 2-fold difference of the observed values. As a result, no statistically significant differences (p > 0.05) were found between most ICE-based and empirical species sensitivity distributions (SSDs). In most cases hazard concentrations were within or below the 95% confidence intervals of the direct-empirical SSD-based values, regardless of model choice. Interspecies correlation estimation-based 5th percentile (HC5) values showed a 200- to 900-fold increase as the log KOW increased from 2 to 5.3. Results indicate that ICE models for aromatic compounds provide a statistically based approach for deriving conservative hazard estimates for protecting aquatic life. PMID:26184086

  6. Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters

    PubMed Central

    2010-01-01

    Background The vertebrate globin genes encoding the α- and β-subunits of the tetrameric hemoglobins are clustered at two unlinked loci. The highly conserved linear order of the genes flanking the hemoglobins provides a strong anchor for inferring common ancestry of the globin clusters. In fish, the number of α-β-linked globin genes varies considerably between different sublineages and seems to be related to prevailing physico-chemical conditions. Draft sequences of the Atlantic cod genome enabled us to determine the genomic organization of the globin repertoire in this marine species that copes with fluctuating environments of the temperate and Arctic regions. Results The Atlantic cod genome was shown to contain 14 globin genes, including nine hemoglobin genes organized in two unlinked clusters designated β5-α1-β1-α4 and β3-β4-α2-α3-β2. The diverged cod hemoglobin genes displayed different expression levels in adult fish, and tetrameric hemoglobins with or without a Root effect were predicted. The novel finding of maternally inherited hemoglobin mRNAs is consistent with a potential role played by fish hemoglobins in the non-specific immune response. In silico analysis of the six teleost genomes available showed that the two α-β globin clusters are flanked by paralogs of five duplicated genes, in agreement with the proposed teleost-specific duplication of the ancestral vertebrate globin cluster. Screening the genome of extant urochordate and cephalochordate species for conserved globin-flanking genes revealed linkage of RHBDF1, MPG and ARHGAP17 to globin genes in the tunicate Ciona intestinalis, while these genes together with LCMT are closely positioned in amphioxus (Branchiostoma floridae), but seem to be unlinked to the multiple globin genes identified in this species. Conclusion The plasticity of Atlantic cod to variable environmental conditions probably involves the expression of multiple globins with potentially different properties. The

  7. Ligand binding site of tear lipocalin: contribution of a trigonal cluster of charged residues probed by 8-anilino-1-naphthalenesulfonic acid.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2008-02-01

    Human tear lipocalin (TL) exhibits diverse functions, most of which are linked to ligand binding. To map the binding site of TL for some amphiphilic ligands, we capitalized on the hydrophobic and hydrophilic properties of 8-anilino-1-naphthalenesulfonic acid (ANS). In single Trp mutants, resonance energy transfer from Trp to ANS indicates that the naphthalene group of ANS is proximate to Leu105 in the cavity. Binding energies of TL to ANS and its analogues reveal contributions from electrostatic interactions. The sulfonate group of ANS interacts strongly with the nonconserved intracavitary residue Lys114 and less with neighboring residues His84 and Glu34. This trigonal cluster of residues may play a role in the ligand recognition site for some negatively charged ligands. Because many drugs possess sulfonate groups, the trigonal cluster-sulfonate interaction can also be exploited as a lipocalin-based drug delivery mechanism. The binding of lauric acid and its analogues shows that fatty acids assume heterogeneous orientations in the cavity of TL. Predominantly, the hydrocarbon tail is buried in the cavity of TL and the carboxyl group is oriented toward the mouth. However, TL can also interact, albeit relatively weakly, with fatty acids oriented in the opposite direction. As the major lipid binding protein of tears, the ability to accommodate fatty acids in two opposing orientations may have functional implications for TL. At the aqueous-lipid interface, fatty acids whose carboxyl groups are positioned toward the aqueous phase are available for interaction with TL that could augment stability of the tear film. PMID:18179255

  8. Conserved Glycine Residues in the Cytoplasmic Domain of the Aspartate Receptor Play Essential Roles in Kinase Coupling and On–Off Switching†

    PubMed Central

    Coleman, Matthew D.; Bass, Randal B.; Mehan, Ryan S.; Falke, Joseph J.

    2010-01-01

    The aspartate receptor of the bacterial chemotaxis pathway serves as a scaffold for the formation of a multiprotein signaling complex containing the receptor and the cytoplasmic pathway components. Within this complex, the receptor regulates the autophosphorylation activity of histidine kinase CheA, thereby controlling the signals sent to the flagellar motor and the receptor adaptation system. The receptor cytoplasmic domain, which controls the on–off switching of CheA, possesses 14 glycine residues that are highly conserved in related receptors. In principle, these conserved glycines could be required for static turns, bends, or close packing in the cytoplasmic domain, or they could be required for conformational dynamics during receptor on–off switching. To determine which glycines are essential and to probe their functional roles, we have substituted each conserved glycine with both alanine and cysteine, and then measured the effects on receptor function in vivo and in vitro. The results reveal a subset of six glycines which are required for receptor function during cellular chemotaxis. Two of these essential glycines (G388 and G391) are located at a hairpin turn at the distal end of the folded cytoplasmic domain, where they are required for the tertiary fold of the signaling subdomain and for CheA kinase activation. Three other essential glycines (G338, G339, and G437) are located at the border between the adaptation and signaling subdomains, where they play key roles in CheA kinase activation and on–off switching. These three glycines form a ring around the four-helix bundle that comprises the receptor cytoplasmic domain, yielding a novel architectural feature termed a bundle hinge. The final essential glycine (G455) is located in the adaptation subdomain where it is required for on–off switching. Overall, the findings confirm that six of the 14 conserved cytoplasmic glycines are essential for receptor function because they enable helix turns and bends

  9. Evolutionary conservation of the mouse apolipoprotein e-c1-c2 gene cluster: Structure and genetic variability in inbred mice

    SciTech Connect

    Hoffer, M.J.V.; Hofker, M.H.; Eck, M.M. van; Frants, R.R. ); Havekes, L.M. )

    1993-01-01

    The human apolipoprotein E (APOE), APOC1, pseudo APOC1 (APOC1[prime]), and APOC2 genes are clustered within 48 kb on the long arm of chromosome 19. A mouse Apoe cDNA probe was used to isolate overlapping cosmid clones from a cosmid library of the C57BL/Rij inbred mouse strain. These clones were investigated for the presence of the Apocl and Apoc2 genes by heterologous hybridization. Our results show that the Apoe-cl-c2 gene cluster is conserved in the mouse. In line with evolutionary data, the mouse lacks the equivalent of APOC1[prime]. These data were confirmed using a mouse Apoc2 cDNA clone, and surprisingly the CDNA clone isolated here was 965 bp in size, which is on average 450 bp longer than other APOC2 cDNAs described so far. Correspondingly, the Apoc2 gene occupies an unusually large genomic region, due to an extended 5[prime] end. Interestingly, a variable number of tandem repeat (VNTR) in the third intron of the human APOC2 gene shows a high sequence homology and is located at the identical position in the mouse gene. Despite the high copy number of this VNTR (27 or 34 copies) only two variants were found among 11 different inbred strains. With the aid of six restriction fragment length variations in this gene cluster only two different haplotypes could be deduced, indicating that the Apoe-cl-c2 gene cluster is highly conserved in the inbred strains that were studied. 32 refs., 5 figs., 1 tab.

  10. The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability.

    PubMed

    Smolko, Ana; Šupljika, Filip; Martinčić, Jelena; Jajčanin-Jozić, Nina; Grabar-Branilović, Marina; Tomić, Sanja; Ludwig-Müller, Jutta; Piantanida, Ivo; Salopek-Sondi, Branka

    2016-04-01

    Brassica rapa auxin amidohydrolase (BrILL2) participates in the homeostasis of the plant hormones auxins by hydrolyzing the amino acid conjugates of auxins, thereby releasing the free active form of hormones. Herein, the potential role of the two conserved Cys residues of BrILL2 (at sequence positions 139 and 320) has been investigated by using interdisciplinary approaches and methods of molecular biology, biochemistry, biophysics and molecular modelling. The obtained results show that both Cys residues participate in the regulation of enzyme activity. Cys320 located in the satellite domain of the enzyme is mainly responsible for protein stability and regulation of enzyme activity through polymer formation, as has been revealed by enzyme kinetics and differential scanning calorimetry analysis of the BrILL2 wild type and mutants C320S and C139S. Cys139 positioned in the active site of the catalytic domain is involved in the coordination of one Mn(2+) ion of the bimetal center and is crucial for the enzymatic activity. Although the point mutation Cys139 to Ser causes the loss of enzyme activity, it does not affect the metal binding to the BrILL2 enzyme, as has been shown by isothermal titration calorimetry, circular dichroism spectropolarimetry and differential scanning calorimetry data. MD simulations (200 ns) revealed a different active site architecture of the BrILL2C139S mutant in comparison to the wild type enzyme. Additional possible reasons for the inactivity of the BrILL2C139S mutant have been discussed based on MD simulations and MM-PBSA free energy calculations of BrILL2 enzyme complexes (wt and C139S mutant) with IPA-Ala as a substrate. PMID:26959939

  11. Conserved Residues in the N Terminus of Lipin-1 Are Required for Binding to Protein Phosphatase-1c, Nuclear Translocation, and Phosphatidate Phosphatase Activity*

    PubMed Central

    Kok, Bernard P. C.; Skene-Arnold, Tamara D.; Ling, Ji; Benesch, Matthew G. K.; Dewald, Jay; Harris, Thurl E.; Holmes, Charles F. B.; Brindley, David N.

    2014-01-01

    Lipin-1 is a phosphatidate phosphatase in glycerolipid biosynthesis and signal transduction. It also serves as a transcriptional co-regulator to control lipid metabolism and adipogenesis. These functions are controlled partly by its subcellular distribution. Hyperphosphorylated lipin-1 remains sequestered in the cytosol, whereas hypophosphorylated lipin-1 translocates to the endoplasmic reticulum and nucleus. The serine/threonine protein phosphatase-1 catalytic subunit (PP-1c) is a major protein dephosphorylation enzyme. Its activity is controlled by interactions with different regulatory proteins, many of which contain conserved RVXF binding motifs. We found that lipin-1 binds to PP-1cγ through a similar HVRF binding motif. This interaction depends on Mg2+ or Mn2+ and is competitively inhibited by (R/H)VXF-containing peptides. Mutating the HVRF motif in the highly conserved N terminus of lipin-1 greatly decreases PP-1cγ interaction. Moreover, mutations of other residues in the N terminus of lipin-1 also modulate PP-1cγ binding. PP-1cγ binds poorly to a phosphomimetic mutant of lipin-1 and binds well to the non-phosphorylatable lipin-1 mutant. This indicates that lipin-1 is dephosphorylated before PP-1cγ binds to its HVRF motif. Importantly, mutating the HVRF motif also abrogates the nuclear translocation and phosphatidate phosphatase activity of lipin-1. In conclusion, we provide novel evidence of the importance of the lipin-1 N-terminal domain for its catalytic activity, nuclear localization, and binding to PP-1cγ. PMID:24558042

  12. Reduced susceptibility to all neuraminidase inhibitors of influenza H1N1 viruses with haemagglutinin mutations and mutations in non-conserved residues of the neuraminidase

    PubMed Central

    McKimm-Breschkin, Jennifer L.; Williams, Janelle; Barrett, Susan; Jachno, Kim; McDonald, Mandy; Mohr, Peter G.; Saito, Takehiko; Tashiro, Masato

    2013-01-01

    Objectives We characterized human H1N1 influenza isolate A/Hokkaido/15/02, which has haemagglutinin and neuraminidase mutations that reduce drug susceptibility to oseltamivir, zanamivir and peramivir. Methods One wild-type and three mutant viruses were isolated by plaque purification. Viruses were tested in MUNANA-based enzyme assays, cell culture and receptor binding assays. Results Two viruses had a neuraminidase Y155H mutation that reduced susceptibility in the enzyme inhibition assay to all inhibitors by 30-fold to >100-fold. The Y155H mutation reduced plaque size and affected the stability, Km and pH activity profile of the enzyme. In contrast to previous mutants, this neuraminidase demonstrated a slower rate of inhibitor binding in the IC50 kinetics assay. One virus had both the Y155H mutation and a haemagglutinin D225G mutation that rescued the small-plaque phenotype of the Y155H virus and affected receptor binding and drug susceptibility in cell culture and binding assays. We also isolated a third mutant virus, with both neuraminidase V114I and haemagglutinin D225N mutations, which affected susceptibility in the enzyme inhibition assay and receptor binding, respectively, but to lesser extents than the Y155H and D225G mutations. Conclusions Neither Y155 nor V114 is conserved across neuraminidase subtypes. Furthermore, Y155 is not conserved even among avian and swine N1 viruses. Structurally, both residues reside far from the neuraminidase active site. D225 forms part of the receptor binding site of the haemagglutinin. We believe this is the first demonstration of a specific haemagglutinin mutation correlating with reduced drug susceptibility in plaque assays in both Madin Darby Canine Kidney and SIAT cells. PMID:23759505

  13. Molecular mechanism of the enterococcal aminoglycoside 6'-N-acetyltransferase': role of GNAT-conserved residues in the chemistry of antibiotic inactivation.

    PubMed

    Draker, Kari-ann; Wright, Gerard D

    2004-01-20

    The Gram-positive pathogen Enterococcus faecium is intrinsically resistant to aminoglycoside antibiotics due to the presence of a chromosomally encoded aminoglycoside 6'-N-acetyltransferase [AAC(6')-Ii]. This enzyme is a member of the GCN5-related N-acetyltransferase (GNAT) superfamily and is therefore structurally homologous to proteins that catalyze acetyl transfer to diverse acyl-accepting substrates. This study reports the investigation of several potential catalytic residues that are present in the AAC(6')-Ii active site and also conserved in many GNAT enzymes. Site-directed mutagenesis of Glu72, His74, Leu76, and Tyr147 with characterization of the purified site mutants gave valuable information about the roles of these amino acids in acetyl transfer chemistry. More specifically, steady-state kinetic analysis of protein activity, solvent viscosity effects, pH studies, and antibiotic resistance profiles were all used to assess the roles of Glu72 and His74 as potential active site bases, Tyr147 as a general acid, and the importance of the amide NH group of Leu76 in transition-state stabilization. Taken together, our results indicate that Glu72 is not involved in general base catalysis, but is instead critical for the proper positioning and orientation of aminoglycoside substrates in the active site. Similarly, His74 is also not acting as the active site base, with pH studies revealing that this residue must be protonated for optimal AAC(6')-Ii activity. Mutation of Tyr147 was found not to affect the chemical step of catalysis, and our results were not consistent with this residue acting as a general acid. Last, the amide NH group of Leu76 is implicated in important interactions with acetyl-CoA and transition-state stabilization. In summary, the work described here provides important information regarding the molecular mechanism of AAC(6')-Ii catalysis that allows us to contrast our findings with those of other GNAT proteins and to demonstrate that these enzymes

  14. Conserved-residue mutations in Wzy affect O-antigen polymerization and Wzz-mediated chain-length regulation in Pseudomonas aeruginosa PAO1

    NASA Astrophysics Data System (ADS)

    Islam, Salim T.; Huszczynski, Steven M.; Nugent, Timothy; Gold, Alexander C.; Lam, Joseph S.

    2013-12-01

    O antigen (O-Ag) in many bacteria is synthesized via the Wzx/Wzy-dependent pathway in which Wzy polymerizes lipid-linked O-Ag subunits to modal lengths regulated by Wzz. Characterization of 83 site-directed mutants of Wzy from Pseudomonas aeruginosa PAO1 (WzyPa) in topologically-mapped periplasmic (PL) and cytoplasmic loops (CL) verified the functional importance of PL3 and PL5, with the former shown to require overall cationic properties. Essential Arg residues in the RX10G motifs of PL3 and PL5 were found to be conserved in putative homologues of WzyPa, as was the overall sequence homology between these two periplasmic loops in each protein. Amino acid substitutions in CL6 were found to alter Wzz-mediated O-antigen modality, with evidence suggesting that these changes may perturb the C-terminal WzyPa tertiary structure. Together, these data suggest that the catch-and-release mechanism of O-Ag polymerization is widespread among bacteria and that regulation of polymer length is affected by interaction of Wzz with Wzy.

  15. Conserved Distal Loop Residues in the Hsp104 and ClpB Middle Domain Contact Nucleotide-binding Domain 2 and Enable Hsp70-dependent Protein Disaggregation*

    PubMed Central

    DeSantis, Morgan E.; Sweeny, Elizabeth A.; Snead, David; Leung, Eunice H.; Go, Michelle S.; Gupta, Kushol; Wendler, Petra; Shorter, James

    2014-01-01

    The homologous hexameric AAA+ proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis. PMID:24280225

  16. Role of the semi-conserved histidine residue in the light-sensing domain of LitR, a MerR-type photosensory transcriptional regulator.

    PubMed

    Takano, Hideaki; Mise, Kou; Maruyama, Takafumi; Hagiwara, Kenta; Ueda, Kenji

    2016-08-01

    The LitR/CarH protein family transcriptional regulator is a new type of photoreceptor based on the function of adenosyl B12 (AdoB12) as a light-sensitive ligand. Here, we studied a semi-conserved histidine residue (His132) in the light-sensing (AdoB12-binding) domain at the C-terminus of LitR from a thermophilic Gram-negative bacterium, Thermus thermophilus HB27. The in vivo mutation of His132 within LitR caused a reduction in the rate of carotenoid production in response to illumination. BIAcore analysis revealed that the illuminated-LitRH132A possesses high DNA-binding activity compared to the wild-type protein. The subunit structure analysis showed that LitRH132A performed an incomplete subunit dissociation. The ability of LitRH132A to associate with AdoB12 was reduced compared with that of the wild-type protein in an equilibration dialysis experiment. Overall, these results suggest that His132 of LitR is involved in the association with AdoB12 as well as the light-sensitive DNA-binding activity based on oligomer dissociation. PMID:27283316

  17. A WDR Gene Is a Conserved Member of a Chitin Synthase Gene Cluster and Influences the Cell Wall in Aspergillus nidulans

    PubMed Central

    Guerriero, Gea; Silvestrini, Lucia; Obersriebnig, Michael; Hausman, Jean-Francois; Strauss, Joseph; Ezcurra, Inés

    2016-01-01

    WD40 repeat (WDR) proteins are pleiotropic molecular hubs. We identify a WDR gene that is a conserved genomic neighbor of a chitin synthase gene in Ascomycetes. The WDR gene is unique to fungi and plants, and was called Fungal Plant WD (FPWD). FPWD is within a cell wall metabolism gene cluster in the Ascomycetes (Pezizomycotina) comprising chsD, a Chs activator and a GH17 glucanase. The FPWD, AN1556.2 locus was deleted in Aspergillus nidulans strain SAA.111 by gene replacement and only heterokaryon transformants were obtained. The re-annotation of Aspergilli genomes shows that AN1556.2 consists of two tightly linked separate genes, i.e., the WDR gene and a putative beta-flanking gene of unknown function. The WDR and the beta-flanking genes are conserved genomic neighbors localized within a recently identified metabolic cell wall gene cluster in genomes of Aspergilli. The heterokaryons displayed increased susceptibility to drugs affecting the cell wall, and their phenotypes, observed by optical, confocal, scanning electron and atomic force microscopy, suggest cell wall alterations. Quantitative real-time PCR shows altered expression of some cell wall-related genes. The possible implications on cell wall biosynthesis are discussed. PMID:27367684

  18. Ligand Binding Site of Tear Lipocalin: Contribution of a Trigonal Cluster of Charged Residues Probed by 8-Anilino-1-naphthalenesulfonic Acid†

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2010-01-01

    Human tear lipocalin (TL) exhibits diverse functions, most of which are linked to ligand binding. To map the binding site of TL for some amphiphilic ligands, we capitalized on the hydrophobic and hydrophilic properties of 8-anilino-1-naphthalenesulfonic acid (ANS). In single Trp mutants, resonance energy transfer from Trp to ANS indicates that the naphthalene group of ANS is proximate to Leu105 in the cavity. Binding energies of TL to ANS and its analogues reveal contributions from electrostatic interactions. The sulfonate group of ANS interacts strongly with the nonconserved intracavitary residue Lys114 and less with neighboring residues His84 and Glu34. This trigonal cluster of residues may play a role in the ligand recognition site for some negatively charged ligands. Because many drugs possess sulfonate groups, the trigonal cluster–sulfonate interaction can also be exploited as a lipocalin-based drug delivery mechanism. The binding of lauric acid and its analogues shows that fatty acids assume heterogeneous orientations in the cavity of TL. Predominantly, the hydrocarbon tail is buried in the cavity of TL and the carboxyl group is oriented toward the mouth. However, TL can also interact, albeit relatively weakly, with fatty acids oriented in the opposite direction. As the major lipid binding protein of tears, the ability to accommodate fatty acids in two opposing orientations may have functional implications for TL. At the aqueous–lipid interface, fatty acids whose carboxyl groups are positioned toward the aqueous phase are available for interaction with TL that could augment stability of the tear film. PMID:18179255

  19. Conserved residue lysine165 is essential for the ability of O6-alkylguanine-DNA alkyltransferase to react with O6-benzylguanine.

    PubMed Central

    Xu-Welliver, M; Kanugula, S; Loktionova, N A; Crone, T M; Pegg, A E

    2000-01-01

    The role of lysine(165) in the activity of the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O(6)-benzylguanine (BG), was investigated by changing this lysine to all other 19 possibilities. All of these mutants (except for K165T, which could not be tested as it was too poorly active for assay in crude cell extracts) gave BG-resistant AGTs with increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation (ED(50)) from 100-fold (K165A) to 2400-fold (K165F). Lys(165) is a completely conserved residue in AGTs from many species, and all of the mutations at this site also reduced the ability to repair methylated DNA. The least deleterious change was that to arginine, which reduced the rate constant for DNA repair by approx. 2.5-fold. Mutant K165R resembled all of the other mutants in being highly resistant to BG, with an ED(50) value for inactivation by BG>200-fold greater than wild-type. Detailed studies of purified K165A AGT showed that the rate constant for repair and the binding to methylated DNA substrates were reduced by 10-20-fold. Despite this, the K165A mutant AGT was able to protect cells from alkylating agents and this protection was not abolished by BG. These results show that, firstly, lysine at position 165 is needed for optimal activity of AGT towards methylated DNA substrates and is essential for efficient reaction with BG; and second, even if the AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkylating agents. These results raise the possibility that the conservation of Lys(165) is due to the need for AGT activity towards substrates containing more bulky adducts than O(6)-methylguanine. They also suggest that alterations at Lys(165) may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this

  20. Anthrax Toxin Receptor 1 / Tumor Endothelial Marker 8: Mutation of Conserved Inserted Domain Residues Overrides Cytosolic Control of Protective Antigen Binding†

    PubMed Central

    Ramey, Jordan D.; Villareal, Valerie A.; Ng, Charles; Ward, Sabrina; Xiong, Jian-Ping; Clubb, Robert T.; Bradley, Kenneth A.

    2010-01-01

    Anthrax toxin receptor 1 (ANTXR1) / tumor endothelial marker 8 (TEM8) is one of two known proteinaceous cell surface anthrax toxin receptors. A metal ion dependent adhesion site (MIDAS) present in the integrin-like inserted (I) domain of ANTXR1 mediates the binding of the anthrax toxin subunit, protective antigen (PA). Here we provide evidence that single point mutations in the I domain can override regulation of ANTXR1 ligand-binding activity mediated by intracellular signals. A previously reported MIDAS-mutant of ANTXR1 (T118A) was found to retain normal metal ion binding and secondary structure but failed to bind PA, consistent with a locked inactive state. Conversely, mutation of a conserved I domain phenylalanine residue to a tryptophan (F205W) increased the proportion of cell-surface ANTXR1 that bound PA, consistent with a locked active state. Interestingly, the KD and total amount of PA bound by the isolated ANTXR1 I domain was not affected by the F205W mutation, indicating that ANTXR1 is preferentially found in the active state in the absence of inside-out signaling. Circular dichroism (CD) spectroscopy and 1H-15N heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) revealed that structural changes between T118A, F205W and WT I domains were minor despite a greater than 103-fold difference in their abilities to bind toxin. Regulation of toxin binding has important implications for the design of toxin inhibitors and for the targeting of ANTXR1 for anti-tumor therapies. PMID:20690680

  1. The valine and lysine residues in the conserved FxVTxK motif are important for the function of phylogenetically distant plant cellulose synthases.

    PubMed

    Slabaugh, Erin; Scavuzzo-Duggan, Tess; Chaves, Arielle; Wilson, Liza; Wilson, Carmen; Davis, Jonathan K; Cosgrove, Daniel J; Anderson, Charles T; Roberts, Alison W; Haigler, Candace H

    2016-05-01

    Cellulose synthases (CESAs) synthesize the β-1,4-glucan chains that coalesce to form cellulose microfibrils in plant cell walls. In addition to a large cytosolic (catalytic) domain, CESAs have eight predicted transmembrane helices (TMHs). However, analogous to the structure of BcsA, a bacterial CESA, predicted TMH5 in CESA may instead be an interfacial helix. This would place the conserved FxVTxK motif in the plant cell cytosol where it could function as a substrate-gating loop as occurs in BcsA. To define the functional importance of the CESA region containing FxVTxK, we tested five parallel mutations in Arabidopsis thaliana CESA1 and Physcomitrella patens CESA5 in complementation assays of the relevant cesa mutants. In both organisms, the substitution of the valine or lysine residues in FxVTxK severely affected CESA function. In Arabidopsis roots, both changes were correlated with lower cellulose anisotropy, as revealed by Pontamine Fast Scarlet. Analysis of hypocotyl inner cell wall layers by atomic force microscopy showed that two altered versions of Atcesa1 could rescue cell wall phenotypes observed in the mutant background line. Overall, the data show that the FxVTxK motif is functionally important in two phylogenetically distant plant CESAs. The results show that Physcomitrella provides an efficient model for assessing the effects of engineered CESA mutations affecting primary cell wall synthesis and that diverse testing systems can lead to nuanced insights into CESA structure-function relationships. Although CESA membrane topology needs to be experimentally determined, the results support the possibility that the FxVTxK region functions similarly in CESA and BcsA. PMID:26646446

  2. Product PCNPsurv or the "reduced" evaporation residue cross section σER/σfusion for "hot" fusion reactions studied with the dynamical cluster-decay model

    NASA Astrophysics Data System (ADS)

    Chopra, Sahila; Kaur, Arshdeep; Hemdeep, Gupta, Raj K.

    2016-04-01

    The product PCNPsurv of compound nucleus (CN) fusion probability PCN and survival probability Psurv is calculated to determine the reduced evaporation residue cross section σER/σfusion , denoted σERreduced, with (total) fusion cross section σfusion given as a sum of CN-formation cross section σCN and non-CN cross section σnCN for each reaction, where σCN is the sum of evaporation residue cross section σER and fusion-fission cross section σff and σnCN, if not measured, is estimated empirically as the difference between measured and calculated σfusion. Our calculations of PCN and Psurv, based on the dynamical cluster-decay model, were successfully made for some 17 "hot" fusion reactions, forming different CN of mass numbers ACN˜100 -300 , with deformations of nuclei up to hexadecapole deformations and "compact" orientations for both coplanar (Φc=0∘ ) and noncoplanar (Φc≠0∘ ) configurations, using various different nuclear interaction potentials. Interesting variations of σERreduced with CN excitation energy E*, fissility parameter χ , CN mass ACN, and Coulomb parameter Z1Z2 show that, independent of entrance channel, different isotopes of CN, and nuclear interaction potentials used, the dominant quantity in the product is Psurv, which classifies all the studied CN into three groups of weakly fissioning, radioactive, and strongly fissioning superheavy nuclei, with relative magnitudes of σERreduced˜1 , ˜10-6 , and ˜10-11 , which, like for PCN, get further grouped in two dependencies of (i) weakly fissioning and strongly fissioning superheavy nuclei decreasing with increasing E* and (ii) radioactive nuclei increasing with increasing E*.

  3. Structure based alignment and clustering of proteins (STRALCP)

    DOEpatents

    Zemla, Adam T.; Zhou, Carol E.; Smith, Jason R.; Lam, Marisa W.

    2013-06-18

    Disclosed are computational methods of clustering a set of protein structures based on local and pair-wise global similarity values. Pair-wise local and global similarity values are generated based on pair-wise structural alignments for each protein in the set of protein structures. Initially, the protein structures are clustered based on pair-wise local similarity values. The protein structures are then clustered based on pair-wise global similarity values. For each given cluster both a representative structure and spans of conserved residues are identified. The representative protein structure is used to assign newly-solved protein structures to a group. The spans are used to characterize conservation and assign a "structural footprint" to the cluster.

  4. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication.

    PubMed

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V; Mintaev, Ramil R; Alexeevski, Andrei V; Veit, Michael

    2015-12-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

  5. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication

    PubMed Central

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V.; Mintaev, Ramil R.; Alexeevski, Andrei V.; Veit, Michael

    2015-01-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

  6. Effectiveness and Cost of Insecticide-Treated Bed Nets and Indoor Residual Spraying for the Control of Cutaneous Leishmaniasis: A Cluster-Randomized Control Trial in Morocco.

    PubMed

    Faraj, Chafika; Yukich, Joshua; Adlaoui, El Bachir; Wahabi, Rachid; Mnzava, Abraham Peter; Kaddaf, Mustapha; El Idrissi, Abderrahmane Laamrani; Ameur, Btissam; Kleinschmidt, Immo

    2016-03-01

    Cutaneous leishmaniasis (CL) remains an important public health problem in Morocco. A cluster-randomized trial was conducted with the following three study arms: 1) long-lasting insecticide-treated nets (LLINs) plus standard of care environmental management (SoC-EM), 2) indoor residual spraying (IRS) with α-cypermethrin plus SoC-EM, and 3) SoC-EM alone. Incidence of new CL cases by passive and active case detection, sandfly abundance, and cost and cost-effectiveness was compared between study arms over 5 years. Incidence of CL and sandfly abundance were significantly lower in the IRS arm compared with SoC-EM (CL incidence rate ratio = 0.32, 95% confidence interval [CI] = 0.15-0.69, P = 0.005 and sandfly abundance ratio = 0.39, 95% CI = 0.18-0.85, P = 0.022). Reductions in the LLIN arm of the study were not significant, possibly due to poor compliance. IRS was effective and more cost-effective for the prevention of CL in Morocco. PMID:26811431

  7. Effectiveness and Cost of Insecticide-Treated Bed Nets and Indoor Residual Spraying for the Control of Cutaneous Leishmaniasis: A Cluster-Randomized Control Trial in Morocco

    PubMed Central

    Faraj, Chafika; Yukich, Joshua; Adlaoui, El Bachir; Wahabi, Rachid; Mnzava, Abraham Peter; Kaddaf, Mustapha; El Idrissi, Abderrahmane Laamrani; Ameur, Btissam; Kleinschmidt, Immo

    2016-01-01

    Cutaneous leishmaniasis (CL) remains an important public health problem in Morocco. A cluster-randomized trial was conducted with the following three study arms: 1) long-lasting insecticide-treated nets (LLINs) plus standard of care environmental management (SoC-EM), 2) indoor residual spraying (IRS) with α-cypermethrin plus SoC-EM, and 3) SoC-EM alone. Incidence of new CL cases by passive and active case detection, sandfly abundance, and cost and cost-effectiveness was compared between study arms over 5 years. Incidence of CL and sandfly abundance were significantly lower in the IRS arm compared with SoC-EM (CL incidence rate ratio = 0.32, 95% confidence interval [CI] = 0.15–0.69, P = 0.005 and sandfly abundance ratio = 0.39, 95% CI = 0.18–0.85, P = 0.022). Reductions in the LLIN arm of the study were not significant, possibly due to poor compliance. IRS was effective and more cost-effective for the prevention of CL in Morocco. PMID:26811431

  8. Organic weed conrol and cover crop residue integration impacts on weed control, quality, and yield and economics in conservation tillage tomato - A case study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increased use of conservation tillage in vegetable production requires more information be developed on the role of cover crops in weed control, tomato quality and yield. Three conservation-tillage systems utilizing crimson clover, brassica and cereal rye as winter cover crops were compared to ...

  9. Organic weed conrol and cover crop residue integration impacts on weed control, quality, and yield and economics in conservation tillage tomato- A case study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increased use of conservation tillage in vegetable production requires more information be developed on the role of cover crops in weed control, tomato quality and yield. Three conservation-tillage systems utilizing crimson clover, brassica and cereal rye as winter cover crops were compared to ...

  10. Surf5: A gene in the tightly clustered mouse surfeit locus is highly conserved and transcribed divergently from the rpL7A (Surf3) gene

    SciTech Connect

    Garson, K.; Duhig, T.; Armes, N.; Colombo, P.; Fried, M.

    1995-11-20

    The four previously characterized genes (Surf1 to 4) of the mouse Surfeit locus do not share any sequence homology, and the transcription of each gene alternates with respect to its neighbors. Adjacent Surfeit genes are separated by very small distances, and two of the genes overlap at their 3{prime} ends. In this work we have further defined the Surfeit gene cluster by the isolation of Surf5, a fifth gene of the locus, and determination of its relationship to the other Surfeit genes. Surf5 does not share any sequence homology with the four cloned Surfeit genes. The transcription of Surf5 is divergent with respect to its neighbor the Surf3 gene, and the 5{prime} ends of Surf5 and Surf3 are separated by only 159 bp, suggesting the presence of a second bidirectional promoter in the locus. The 3{prime} end of Surf5 maps only 68 bp away from the processed 3{prime} end of a pseudogene. The human and partial chicken Surf5 coding regions show greater than 95% identity, and a Caenorhabditis elegans homologue shows 38% identity and 56% similarity with the mouse Surf5 amino acid sequence. The 3.5-kb transcript of Surf5 encodes a small hydrophilic protein of 140 amino acid residues, which differs from the ribosomal protein L7a encoded by the Surf3 gene or the integral membrane protein encoded by the Surf4 gene. Subcellular fractionation located the Surf5 protein to the soluble fraction of the cytoplasm. The Surfeit locus appears to represent a novel type of gene cluster in which the genes are unrelated by sequence or function; however, their organization may play a role in their gene expression. 44 refs., 5 figs.

  11. Biochemical characterization of the water-soluble squalene synthase from Methylococcus capsulatus and the functional analyses of its two DXXD(E)D motifs and the highly conserved aromatic amino acid residues.

    PubMed

    Ohtake, Kana; Saito, Naoki; Shibuya, Satoshi; Kobayashi, Wakako; Amano, Ryosuke; Hirai, Takumi; Sasaki, Shinji; Nakano, Chiaki; Hoshino, Tsutomu

    2014-12-01

    Information regarding squalene synthases (SQSs) from prokaryotes is scarce. We aimed to characterize the SQS from Methylococcus capsulatus. We studied its reaction mechanism by kinetic analysis and evaluated the structure of the substrate/inhibitor-binding sites via homology modeling. The cloned M. capsulatus SQS was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid column chromatography. Interestingly, M. capsulatus SQS was water-soluble and did not require any detergent for its higher activity, unlike other SQSs studied previously; supplementation of any type of detergent inhibited enzyme activity. The specific activity and the kinetic values (Km and kcat ) for the substrate farnesyl diphosphate and NADPH are reported. The substrate analog farnesyl methylenediphosphonate showed potent inhibition toward the enzyme. We prepared the site-specific mutants directed at potential active-site residues (58) DXX(61) E(62) D (S1 site) and (213) DXX(216) D(217) D (S2 site), which were assumed to be involved in the binding of the substrate farnesyl diphosphate through the Mg(2+) ion. We first demonstrated that the S1 site and the two basic residues (R55 and K212) were responsible for the binding of farnesyl diphosphate. Furthermore, we examined the catalytic roles of the highly conserved aromatic residues and demonstrated that the Y164 residue abstracts the proton of cation 5, which is produced during the first half-reaction (Scheme 1), to afford presqualene diphosphate, and that the W224 residue stabilizes the intermediary cation 5 via the cation-π interaction. Furthermore, we confirm for the first time that the F32 and the Y51 residues also stabilize the carbocation intermediate(s) generated during the second half-reaction. PMID:25283713

  12. Characterization of G protein coupling mediated by the conserved D1343.49 of DRY motif, M2416.34, and F2516.44 residues on human CXCR1

    PubMed Central

    Han, Xinbing; Feng, Yan; Chen, Xinhua; Gerard, Craig; Boisvert, William A.

    2015-01-01

    CXCR1, a receptor for interleukin-8 (IL-8), plays an important role in defending against pathogen invasion during neutrophil-mediated innate immune response. Human CXCR1 is a G protein-coupled receptor (GPCR) with its characteristic seven transmembrane domains (TMs). Functional and structural analyses of several GPCRs have revealed that conserved residues on TM3 (including the highly conserved Asp-Arg-Tyr (DRY) motif) and TM6 near intracellular loops contain domains critical for G protein coupling as well as GPCR activation. The objective of this study was to elucidate the role of critical amino acid residues on TM3 near intracellular loop 2 (i2) and TM6 near intracellular loop 3 (i3), including S1323.47 (Baldwin location), D1343.49, M2416.34, and F2516.44, in G protein coupling and CXCR1 activation. The results demonstrate that mutations of D1343.49 at DRY motif of CXCR1 (D134N and D134V) completely abolished the ligand binding and functional response of the receptor. Additionally, point mutations at positions 241 and 251 between TM6 and i3 loop generated mutant receptors with modest constitutive activity via Gα15 signaling activation. Our results show that D1343.49 on the highly conserved DRY motif has a distinct role for CXCR1 compared to its homologues (CXCR2 and KSHV-GPCR) in G protein coupling and receptor activation. In addition, M2416.34 and F2516.44 along with our previously identified V2476.40 on TM6 are spatially located in a “hot spot” likely essential for CXCR1 activation. Identification of these amino acid residues may be useful for elucidating mechanism of CXCR1 activation and designing specific antagonists for the treatment of CXCR1-mediated diseases. PMID:25834784

  13. Substitution of conserved cysteine residues in Wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Substitutions in the amino-terminal region of Wheat streak mosaic virus (WSMV) HC-Pro were evaluated for effects on transmission by the wheat curl mite (Aceria tosichella Keifer). Alanine substitution at cysteine residues 16, 46 and 49 abolished vector transmission. Although alanine substitution a...

  14. Sequence-Based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families

    PubMed Central

    Maimanakos, Janine; Chow, Jennifer; Gaßmeyer, Sarah K.; Güllert, Simon; Busch, Florian; Kourist, Robert; Streit, Wolfgang R.

    2016-01-01

    Arylmalonate Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta-, and Gamma-proteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the tripartite tricarboxylate transporters family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99%) of the (R)-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes. PMID:27610105

  15. Sequence-Based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families.

    PubMed

    Maimanakos, Janine; Chow, Jennifer; Gaßmeyer, Sarah K; Güllert, Simon; Busch, Florian; Kourist, Robert; Streit, Wolfgang R

    2016-01-01

    Arylmalonate Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica's prototype appeared to be limited to the classes of Alpha-, Beta-, and Gamma-proteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the tripartite tricarboxylate transporters family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99%) of the (R)-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes. PMID:27610105

  16. Mutation of a Conserved Active Site Residue Converts Tyrosyl-DNA Phosphodiesterase l Into a DNA Topoisomerase l-Dependent Poison

    SciTech Connect

    He,X.; van Waardenburg, R.; Babaoglu, K.; Price, A.; Nitiss, K.; Nitiss, J.; Bjornsti, M.; White, S.

    2007-01-01

    Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 {angstrom} crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H{sub 432}R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H{sub 432}N was drug-independent, coinciding with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H432 mutants were recessive to wild-type Tdp1. Thus, yeast H{sub 432} acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3' phosphotyrosyl and 3' phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H{sub 432}N-catalyzed resolution of 3' phospho-adducts.

  17. Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

    PubMed Central

    Schmid, S R; Linder, P

    1991-01-01

    The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products. Images PMID:2046664

  18. A conserved glutamate residue in the C-terminal deaminase domain of pentatricopeptide repeat proteins is required for RNA editing activity.

    PubMed

    Hayes, Michael L; Dang, Kim N; Diaz, Michael F; Mulligan, R Michael

    2015-04-17

    Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions. The glutamate residue of the HXE motif is catalytically required in the reaction catalyzed by cytidine deaminase. In this work, we examine the activity of the DYW deaminase domain through truncation or mutagenesis of the HXE motif. OTP84 is required for editing three chloroplast sites, and transgenes expressing OTP84 with C-terminal truncations were capable of editing only one of the three cognate sites at high efficiency. These results suggest that the deaminase domain of OTP84 is required for editing two of the sites, but another deaminase is able to supply the deamination activity for the third site. OTP84 and CREF7 transgenes were mutagenized to replace the glutamate residue of the HXE motif, and transgenic plants expressing OTP84-E824A and CREF7-E554A were unable to efficiently edit the cognate editing sites for these genes. In addition, plants expressing CREF7-E554A exhibited substantially reduced capacity to edit a non-cognate site, rpoA C200. These results indicate that the DYW deaminase domains of PPR proteins are involved in editing their cognate editing sites, and in some cases may participate in editing additional sites in the chloroplast. PMID:25739442

  19. Conserved water mediated H-bonding dynamics of Ser117 and Thr119 residues in human transthyretin-thyroxin complexation: inhibitor modeling study through docking and molecular dynamics simulation.

    PubMed

    Banerjee, Avik; Bairagya, Hridoy R; Mukhopadhyay, Bishnu P; Nandi, Tapas K; Mishra, Deepak K

    2013-07-01

    Transthyretin (TTR) is a protein whose aggregation and deposition causes amyloid diseases in human beings. Amyloid fibril formation is prevented by binding of thyroxin (T4) or its analogs to TTR. The MD simulation study of several solvated X-ray structures of apo and holo TTR has indicated the role of a conserved water molecule and its interaction with T4 binding residues Ser117 and Thr119. Geometrical and electronic consequences of those interactions have been exploited to design a series of thyroxin analogs (Mod1-4) by modifying 5' or 3' or both the iodine atoms of thyroxin. Binding energy of the designed ligands has been calculated by docking the molecules in tetrameric structure of the protein. Theoretically investigated pharmacological parameters along with the binding energy data indicate the potentiality of 3',5'-diacetyl-3,5-dichloro-l-thyronine (Mod4) to act as a better inhibitor for TTR-related amyloid diseases. PMID:23732306

  20. Amino acid substitutions of conserved residues in the carboxyl-terminal domain of the [alpha]I(X) chain of type X collagen occur in two unrelated families with metaphyseal chondrodysplasia type Schmid

    SciTech Connect

    Wallis, G.A.; Rash, B.; Sweetman, W.A.; Thomas, J.T.; Grant, M.E.; Boot-Handford, R.P. ); Super, M. ); Evans, G. )

    1994-02-01

    Type X collagen is a homotrimeric, short-chain, nonfibrillar extracellular-matrix component that is specifically and transiently synthesized by hypertrophic chondrocytes at the site of endochondral ossification. The precise function of type X collagen is not known, but its specific pattern of expression suggests that mutations within the encoding gene (COL10A1) that alter the structure or synthesis of the protein may cause heritable forms of chondrodysplasia. The authors used the PCR and the SSCP techniques to analyze the coding and upstream promoter regions of the COL10A1 gene in a number of individuals with forms of chondrodysplasia. Using this approach, they identified two individuals with metaphyseal chondrodysplasia type Schmid (MCDS) with SSCP changes in the region of the gene encoding the carboxyl-terminal domain. Sequence analysis demonstrated that the individuals were heterozygous for two unique single-base-pair transitions that led to the substitution of the highly conserved amino acid residue tyrosine at position 598 by aspartic acid in one person and of leucine at position 614 by proline in the other. The substitution at residue 598 segregated with the phenotype in a family of eight (five affected and three unaffected) related persons. The substitutions at residue 614 occurred in a sporadically affected individual but not in her unaffected mother and brother. Additional members of this family were not available for further study. These results suggest that certain amino acid substitutions within the carboxyl-terminal domain of the chains of the type X collagen molecule cause MCDS. These amino acid substitutions are likely to alter either chain recognition or assembly of the type X collagen molecule, thereby depleting the amount of normal type X collagen deposited in the extracellular matrix, with consequent aberrations in bone growth and development. 36 refs., 5 figs.

  1. A differentially conserved residue (Ile42) of GH42 β-galactosidase from Geobacillus stearothermophilus BgaB is involved in both catalysis and thermostability.

    PubMed

    Dong, Yi-Ning; Chen, Hai-Qin; Sun, Yan-Hui; Zhang, Hao; Chen, Wei

    2015-04-01

    The glycoside hydrolase family 42 (GH42) of thermophilic microorganisms consists of thermostable β-galactosidases that display significant variations in their temperature optima and stabilities. In this study, we compared the substrate binding modes of 2 GH42 β-galactosidases, BgaB from Geobacillus stearothermophilus and A4-β-Gal from Thermus thermophilus A4. The A4-β-Gal has a catalytic triad (Glu312-Arg32-Glu35) with an extended hydrogen bond network that has not been observed in BgaB. In this study, we performed site-saturation mutagenesis of Ile42 in BgaB (equivalent to Glu312 in A4-β-Gal) to study the effects of different residues on thermostability, catalytic function, and the extended hydrogen bond network. Our experimental results suggest that substitution of Ile42 with polar AA enhanced the thermostability but decreased the catalytic efficiency of BgaB. Polar AA substitution for Ile42 simultaneously affected thermostability, catalytic efficiency, and the hydrogen bond network, suggesting that Ile42 is responsible for functional discrimination between members of the GH42 family. These observations could lead to a novel strategy for investigating the functional evolution of the GH42 β-galactosidases. PMID:25682138

  2. Conserved residues of the Pro103–Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase

    PubMed Central

    Hill, Benjamin L; Wong, Jennifer; May, Brian M; Huerta, Fidel B; Manley, Tara E; Sullivan, Peter RF; Olsen, Kenneth W; Ballicora, Miguel A

    2015-01-01

    The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteric signal in the Escherichia coli ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize that a Pro103–Arg115 loop is part of an activation path. It had strongly correlated movements with regions of the enzyme associated with regulation and ATP binding, and a network analysis showed that the optimal network pathways linking ATP and the activator binding Lys39 mainly involved residues of this loop. This hypothesis was biochemically tested by mutagenesis. We found that several alanine mutants of the Pro103–Arg115 loop had altered activation profiles for fructose-1,6-bisphosphate. Mutants P103A, Q106A, R107A, W113A, Y114A, and R115A had the most altered kinetic profiles, primarily characterized by a lack of response to fructose-1,6-bisphosphate. This loop is a distinct insertional element present only in allosterically regulated sugar nucleotide pyrophosphorylases that could have been acquired to build a triggering mechanism to link proto-allosteric and catalytic sites. PMID:25620658

  3. Aminoacetylation Reaction Catalyzed by Leucyl-tRNA Synthetase Operates via a Self-Assisted Mechanism Using a Conserved Residue and the Aminoacyl Substrate.

    PubMed

    Aleksandrov, Alexey; Palencia, Andrés; Cusack, Stephen; Field, Martin

    2016-05-19

    Leucyl-tRNA synthetase catalyzes attachment of leucine amino acid to its cognate tRNA. During the second, aminoacetylation, step of the reaction, the leucyl moiety is transferred from leucyl-adenylate to the terminal A76 adenosine of tRNA. In this work, we have investigated the aminoacetylation step catalyzed by leucyl-tRNA synthase, using ab initio quantum chemical/molecular mechanical hybrid potentials in conjunction with reaction-path-location algorithms and molecular dynamics free energy simulations. We have modeled reaction mechanisms arising from both crystallographic studies and computational work. We invoke various groups as potential proton acceptors-namely, the phosphate and leucyl amino groups of leucyl-adenylate, the A76 base of tRNA, and the Asp80 and Glu532 residues of the protein-and consider both metal-assisted and metal-free reactions. Free energy calculations indicate that both the phosphate group of leucyl adenylate and Glu532 are not strong bases. This agrees with the results of the quantum chemical/molecular mechanical reaction path calculations which give high free energy barriers for the studied pathways involving these groups. A self-assisted mechanism with the leucyl amino group and Asp80 as proton acceptors is the most likely. Furthermore, in this mechanism the presence of a metal ion coordinated by the phosphate group and Glu532 strongly activates the reaction. PMID:27115861

  4. Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

    PubMed Central

    Bertrand, L; Vertommen, D; Feytmans, E; Di Pietro, A; Rider, M H; Hue, L

    1997-01-01

    Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis. PMID:9032444

  5. Mutational and Structural Analysis of Conserved Residues in Ribose-5-Phosphate Isomerase B from Leishmania donovani: Role in Substrate Recognition and Conformational Stability

    PubMed Central

    Kaur, Preet Kamal; Tripathi, Neha; Desale, Jayesh; Neelagiri, Soumya; Yadav, Shailendra; Bharatam, Prasad V.; Singh, Sushma

    2016-01-01

    Ribose-5-phosphate isomerase B from Leishmania donovani (LdRpiB) is one of the potential drug targets against visceral leishmaniasis. In the present study, we have targeted several conserved amino acids for mutational analysis (i.e. Cys69, His11, His102, His138, Asp45, Tyr46, Pro47 and Glu149) to gain crucial insights into their role in substrate binding, catalysis and conformational stability of the enzyme. All the eight LdRpiB variants were cloned, sequenced, expressed and purified. C69S, H102N, D45N and E149A mutants exhibited complete loss of enzyme activity indicating that they are indispensable for the enzyme activity. Kinetic parameters were altered in case of H138N, H11N and P47A variants; however Y46F exhibited similar kinetic behaviour as wild type. All the mutants except H138N exhibited altered protein structure as determined by CD and fluorescence spectral analysis. This data was supported by the atomic level details of the conformational changes and substrate binding using molecular dynamic simulations. LdRpiB also exhibited activity with D-form of various aldose substrates in the order of D-ribose > D-talose > D-allose > D-arabinose. Our study provides insights for better understanding of substrate enzyme interactions which can rationalize the process of drug design against parasite RpiB. PMID:26953696

  6. Identification of a cluster of residues in transmembrane segment 6 of domain III of the cockroach sodium channel essential for the action of pyrethroid insecticides

    PubMed Central

    Du, Yuzhe; Lee, Jung-Eun; Nomura, Yoshiko; Zhang, Tianxiang; Zhorov, Boris S.; Dong, Ke

    2011-01-01

    A phenylalanine residue (Phe1519) in the sixth transmembrane segment of domain III (IIIS6) of the cockroach BgNav sodium channel is required for the binding and action of pyrethroids. However, whether or not other residues in IIIS6 participate in the action of pyrethroids remains to be determined. In the present study, we conducted a systematic analysis of 20 residues in IIIS6 of the BgNav channel using alanine-scanning mutagenesis. Our results show that alanine substitutions of four residues, Ile1514, Gly1516, Phe1518 and Asn1522, altered sodium channel sensitivity to pyrethroid insecticides. Whereas the G1516A, F1518A and N1522A substitutions diminished sodium channel sensitivity to all seven pyrethroids examined, including four type I (lacking the α-cyano group at the phenoxybenzyl alcohol) and three type II (containing the α-cyano group) pyrethroids, the I1514A substitution enhanced sodium channel sensitivity to four type I and type II pyrethroids that contain the phenoxybenzyl alcohol only. We also show that alanine/lysine substitutions of Leu1521 and Ser1517 affected the action of BTX (batrachotoxin), but not pyrethroids. In the Kv1.2-based homology model of the open sodium channel, side chains of Ile1514, Phe1518 and Asn1522 are exposed towards helix IIS5 and linker IIS4–IIS5, which contain previously identified pyrethroid-interacting residues, whereas Ser1517 and Leu1521 face the inner pore where the BTX receptor is located. Thus the present study provides further evidence for structural models in which pyrethroids bind to the lipid-exposed interface formed by helices IIIS6, IIS5 and linker helix IIS4–IIS5, whereas BTX binds to the pore-exposed side of the IIIS6 helix. PMID:19154185

  7. Although divergent in residues of the peptide binding site, conserved chimpanzee Patr-AL and polymorphic human HLA-A*02 have overlapping peptide-binding repertoires.

    PubMed

    Gleimer, Michael; Wahl, Angela R; Hickman, Heather D; Abi-Rached, Laurent; Norman, Paul J; Guethlein, Lisbeth A; Hammond, John A; Draghi, Monia; Adams, Erin J; Juo, Sean; Jalili, Roxana; Gharizadeh, Baback; Ronaghi, Mostafa; Garcia, K Christopher; Hildebrand, William H; Parham, Peter

    2011-02-01

    Patr-AL is an expressed, non-polymorphic MHC class I gene carried by ∼50% of chimpanzee MHC haplotypes. Comparing Patr-AL(+) and Patr-AL(-) haplotypes showed Patr-AL defines a unique 125-kb genomic block flanked by blocks containing classical Patr-A and pseudogene Patr-H. Orthologous to Patr-AL are polymorphic orangutan Popy-A and the 5' part of human pseudogene HLA-Y, carried by ∼10% of HLA haplotypes. Thus, the AL gene alternatively evolved in these closely related species to become classical, nonclassical, and nonfunctional. Although differing by 30 aa substitutions in the peptide-binding α(1) and α(2) domains, Patr-AL and HLA-A*0201 bind overlapping repertoires of peptides; the overlap being comparable with that between the A*0201 and A*0207 subtypes differing by one substitution. Patr-AL thus has the A02 supertypic peptide-binding specificity. Patr-AL and HLA-A*0201 have similar three-dimensional structures, binding peptides in similar conformation. Although comparable in size and shape, the B and F specificity pockets of Patr-AL and HLA-A*0201 differ in both their constituent residues and contacts with peptide anchors. Uniquely shared by Patr-AL, HLA-A*0201, and other members of the A02 supertype are the absence of serine at position 9 in the B pocket and the presence of tyrosine at position 116 in the F pocket. Distinguishing Patr-AL from HLA-A*02 is an unusually electropositive upper face on the α(2) helix. Stimulating PBMCs from Patr-AL(-) chimpanzees with B cells expressing Patr-AL produced potent alloreactive CD8 T cells with specificity for Patr-AL and no cross-reactivity toward other MHC class I molecules, including HLA-A*02. In contrast, PBMCs from Patr-AL(+) chimpanzees are tolerant of Patr-AL. PMID:21209280

  8. A Conserved Active Site Tyrosine Residue of Proline Dehydrogenase Helps Enforce the Preference for Proline over Hydroxyproline as the Substrate†,‡

    PubMed Central

    Ostrander, Elizabeth L.; Larson, John D.; Schuermann, Jonathan P.; Tanner, John J.

    2009-01-01

    Proline dehydrogenase (PRODH) catalyzes the oxidation of L-proline to Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-L-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insights into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analog L-tetrahydro-2-furoic acid were determined at resolutions of 1.75 Å, 1.90 Å and 1.85 Å. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline three-fold and decreases the specificity for proline by factors of twenty (Y540S) and fifty (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate. PMID:19140736

  9. The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6*

    PubMed Central

    de la Roche, Jeanne; Eberhardt, Mirjam J.; Klinger, Alexandra B.; Stanslowsky, Nancy; Wegner, Florian; Koppert, Wolfgang; Reeh, Peter W.; Lampert, Angelika; Fischer, Michael J. M.; Leffler, Andreas

    2013-01-01

    The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons. PMID:23709225

  10. Grouping of amino acids and recognition of protein structurally conserved regions by reduced alphabets of amino acids.

    PubMed

    Li, Jing; Wang, Wei

    2007-06-01

    Sequence alignment is a common method for finding protein structurally conserved/similar regions. However, sequence alignment is often not accurate if sequence identities between to-be-aligned sequences are less than 30%. This is because that for these sequences, different residues may play similar structural roles and they are incorrectly aligned during the sequence alignment using substitution matrix consisting of 20 types of residues. Based on the similarity of physicochemical features, residues can be clustered into a few groups. Using such simplified alphabets, the complexity of protein sequences is reduced and at the same time the key information encoded in the sequences remains. As a result, the accuracy of sequence alignment might be improved if the residues are properly clustered. Here, by using a database of aligned protein structures (DAPS), a new clustering method based on the substitution scores is proposed for the grouping of residues, and substitution matrices of residues at different levels of simplification are constructed. The validity of the reduced alphabets is confirmed by relative entropy analysis. The reduced alphabets are applied to recognition of protein structurally conserved/similar regions by sequence alignment. The results indicate that the accuracy or efficiency of sequence alignment can be improved with the optimal reduced alphabet with N around 9. PMID:17609897