Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P.

PubMed

Zhang, Xiaoxiao; Farah, Nadya; Rolston, Laura; Ericsson, Daniel J; Catanzariti, Ann-Maree; Bernoux, Maud; Ve, Thomas; Bendak, Katerina; Chen, Chunhong; Mackay, Joel P; Lawrence, Gregory J; Hardham, Adrienne; Ellis, Jeffrey G; Williams, Simon J; Dodds, Peter N; Jones, David A; Kobe, Bostjan

2018-05-01

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector-triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc-finger-like structure with a novel interleaved zinc-binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P-mediated recognition. The first zinc-coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid-binding and chromatin-associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non-conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition. © 2017 BSPP AND JOHN WILEY & SONS LTD.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruel, Nancy; Zago, Anna; Spear, Patricia G.

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 mutantmore » 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.« less

A proposed OB-fold with a protein-interaction surface in Candida albicans telomerase protein Est3

PubMed Central

Yu, Eun Young; Wang, Feng; Lei, Ming; Lue, Neal F

2008-01-01

Ever shorter telomeres 3 (Est3) is an essential telomerase regulatory subunit thought to be unique to budding yeasts. Here we use multiple sequence alignment and hidden Markov model–hidden Markov model (HMM-HMM) comparison to uncover potential similarities between Est3 and the mammalian telomeric protein Tpp1. Analysis of site-specific mutants of Candida albicans Est3 revealed functional distinctions between residues that are conserved between Est3 and Tpp1 and those that are unique to Est3. Although both types of residues are important for telomere maintenance in vivo, only the former contributes to telomerase activity in vitro and facilitates the association of Est3 with telomerase core components. Consistent with a function in protein-protein interaction, the residues common to Est3 and Tpp1 map to one face of an OB-fold model structure, away from the canonical nucleic acid binding surface. We propose that Est3 and the OB-fold domain of Tpp1 mediate a conserved function in telomerase regulation. PMID:19172753

AmeriFlux US-Ne1 Mead - irrigated continuous maize site

DOE Data Explorer

Suyker, Andy [University of Nebraska - Lincoln

2016-01-01

This is the AmeriFlux version of the carbon flux data for the site US-Ne1 Mead - irrigated continuous maize site. Site Description - The study site is one of three fields (all located within 1.6 km of each other) at the University of Nebraska Agricultural Research and Development Center near Mead, Nebraska. This site is irrigated with a center pivot system. Prior to the initiation of the study, the irrigated site had a 10-yr history of maize-soybean rotation under no-till. A tillage operation (disking) was done just prior to the 2001 planting to homogenize the top 0.1 m of soil, incorporate P and K fertilizers, as well as previously accumulated surface residues. Since the tillage operation, the site has been under no-till management until the harvest of 2005. Following harvest, a conservation-plow tillage operation was initiated where a small amount of N fertilizer is sprayed on the residue immediately prior to the plow operation. Approximately 1/3 of the crop residue is left on the surface. The post-harvest conservation-plow operation continues as the current practice.

Evolutionarily Conserved Linkage between Enzyme Fold, Flexibility, and Catalysis

PubMed Central

Ramanathan, Arvind; Agarwal, Pratul K.

2011-01-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme–substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme–substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design. PMID:22087074

Evolutionarily conserved linkage between enzyme fold, flexibility, and catalysis.

PubMed

Ramanathan, Arvind; Agarwal, Pratul K

2011-11-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme-substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme-substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.

Base of the Measles Virus Fusion Trimer Head Receives the Signal That Triggers Membrane Fusion*

PubMed Central

Apte-Sengupta, Swapna; Negi, Surendra; Leonard, Vincent H. J.; Oezguen, Numan; Navaratnarajah, Chanakha K.; Braun, Werner; Cattaneo, Roberto

2012-01-01

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses. PMID:22859308

A deeper analysis of the epitope/paratope of PLY-5, a mouse monoclonal antibody which recognises the conserved undecapeptide tryptophan-rich loop (ECTGLAWEWWR) of bacterial cholesterol-dependent cytolysins.

PubMed

González-Menéndez, Pedro; García-Ocaña, Marcos; de los Toyos, Juan R

2013-01-04

A previous study showed that the minimal epitope recognised by the PLY-5 mAb in the conserved undecapeptide Trp-rich loop of bacterial CDCs should consist of WEWWRT (Jacobs et al., 1999) [5]. Now, through immunoscreening of amino acid substitution analogues, it is concluded that the second Trp and the Arg residues are essential in the PLY-5 epitope. The E residue is an auxiliary epitope contributor. Antibody modelling and docking simulations provided support for these findings. For recognition by the antibody, the Trp-rich loop flipped out, mimicking the mechanism of membrane insertion. The displaced second Trp was seen to establish aromatic stacking interactions with aromatic residues of the antibody paratope and the notably extruded guanidium tip of the arginine residue mediated electrostatic interactions with well-exposed carboxylic groups of glutamic residues on the surface of the paratope. Thus, the epitope/paratope interaction is mainly mediated by aromatic and by ionic interactions. Copyright © 2012 Elsevier Inc. All rights reserved.

Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Germane, Katherine L.; Spiller, Benjamin W.

2011-09-20

Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

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

Analysis of the ionic interaction between the hydrophobin RodA and two cutinases of Aspergillus nidulans obtained via an Aspergillus oryzae expression system.

PubMed

Tanaka, Takumi; Nakayama, Mayumi; Takahashi, Toru; Nanatani, Kei; Yamagata, Youhei; Abe, Keietsu

2017-03-01

Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. In the fungus Aspergillus oryzae, the hydrophobin RolA and the polyesterase CutL1 are co-expressed when the sole available carbon source is the biodegradable polyester polybutylene succinate-co-adipate (PBSA). RolA promotes the degradation of PBSA by attaching to the particle surface, changing its structure and interacting with CutL1 to concentrate CutL1 on the PBSA surface. We previously reported that positively charged residues in RolA and negatively charged residues in CutL1 are cooperatively involved in the ionic interaction between RolA and CutL1. We also reported that hydrophobin RodA of the model fungus Aspergillus nidulans, which was obtained via an A. oryzae expression system, interacted via ionic interactions with CutL1. In the present study, phylogenetic and alignment analyses revealed that the N-terminal regions of several RolA orthologs contained positively charged residues and that the corresponding negatively charged residues on the surface of CutL1 that were essential for the RolA-CutL1 interaction were highly conserved in several CutL1 orthologs. A PBSA microparticle degradation assay, a pull-down assay using a dispersion of Teflon particles, and a kinetic analysis using a quartz crystal microbalance revealed that recombinant A. nidulans RodA interacted via ionic interactions with two recombinant A. nidulans cutinases. Together, these results imply that ionic interactions between hydrophobins and cutinases may be common among aspergilli and other filamentous fungi.

Conserved water mediated recognition and the dynamics of active site Cys 331 and Tyr 411 in hydrated structure of human IMPDH-II.

PubMed

Bairagya, Hridoy R; Mukhopadhyay, Bishnu P; Bera, Asim K

2011-01-01

Inosine monophosphate dehydrogenase (IMPDH) of human is involved in GMP biosynthesis pathway, increased level of IMPDH-II (an isoform of enzyme) activity have found in leukemic and sarcoma cells. Modeling and extensive molecular dynamics simulation (15 ns) studies of IMPDH-II (1B3O PDB structure) have indicated the intricate involvement of four conserved water molecules (W 1, W 2, W 3, and W 4) in the conformational transition or the mobilities of "flap" (residues 400-450) and "loop" (residues 325-342) regions in enzyme. The stabilization of active site residues Asn 303, Gly 324, Ser 329, Cys 331, Asp 364, and Tyr 411 through variable H-bonding coordination from the conserved water molecular center seems interesting in the uninhibited hydrated form of human IMPDH-II structures. This conformational transition or the flexibility of mobile regions, water molecular recognition to active site residues Cys 331 and Tyr 411, and the presence of a hydrophilic cavity approximately 540 Å(3) (enclaved by the loop and flap region) near the C-terminal surface of this enzyme may explore a rational hope toward the water mimic inhibitor or anticancer agent design for human. 2010 John Wiley & Sons, Ltd.

Crystal Structure of VC0702 at 2.0 angstrom: A Conserved Hypothetical Protein from Vibrio Cholerae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ni, Shuisong; Forouhar, Farhad; Bussiere, Dirksen E.

2006-06-01

VC0702, a conserved hypothetical protein of unknown function from Vibrio cholerae, resides in a putative three-gene operon containing the MbaA gene, which is involved in regulating formation of the extracellular matrix of biofilms in Vibrio cholerae. The VC0702 crystal structure has been determined at 2.0? and refined to Rwork=22.8% and Rfree=26.3%. VC0702 crystallized in an orthorhombic crystal lattice in the C2221 space group with dimensions of a=66.61 ?, b=88.118 ?, and c=118.35 ? with a homodimer in the asymmetric unit. VC0702 belongs to the Pfam DUF84 and COG1986 family of proteins. Sequence conservation within the DUF84 and COG1986 families wasmore » used to identify a conserved patch of surface residues that define a cleft and potential substrate-binding site in VC0702. The three-dimensional structure of VC0702 is similar to that of Mj0226 from Methanococcus janeshii, which has been identified as a novel NTPase. The NTP-binding site in Mj0226 is similarly located in comparison to the conserved patch of surface residues in VC0702. Furthermore, the NTP binds to MJ0226 in a cleft and deep cavity, features that are present in the VC0702 structure as well, suggesting that VC0702 may have a biochemical function involving NTP binding that is associated with a cellular function of regulating biofilm formation in Vibrio cholerae.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramanathan, Arvind; Agarwal, Pratul K

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function.more » Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.« less

Conserved Glycine Residues in the Fusion Peptide of the Paramyxovirus Fusion Protein Regulate Activation of the Native State

PubMed Central

Russell, Charles J.; Jardetzky, Theodore S.; Lamb, Robert A.

2004-01-01

Hydrophobic fusion peptides (FPs) are the most highly conserved regions of class I viral fusion-mediating glycoproteins (vFGPs). FPs often contain conserved glycine residues thought to be critical for forming structures that destabilize target membranes. Unexpectedly, a mutation of glycine residues in the FP of the fusion (F) protein from the paramyxovirus simian parainfluenza virus 5 (SV5) resulted in mutant F proteins with hyperactive fusion phenotypes (C. M. Horvath and R. A. Lamb, J. Virol. 66:2443-2455, 1992). Here, we constructed G3A and G7A mutations into the F proteins of SV5 (W3A and WR isolates), Newcastle disease virus (NDV), and human parainfluenza virus type 3 (HPIV3). All of the mutant F proteins, except NDV G7A, caused increased cell-cell fusion despite having slight to moderate reductions in cell surface expression compared to those of wild-type F proteins. The G3A and G7A mutations cause SV5 WR F, but not NDV F or HPIV3 F, to be triggered to cause fusion in the absence of coexpression of its homotypic receptor-binding protein hemagglutinin-neuraminidase (HN), suggesting that NDV and HPIV3 F have stricter requirements for homotypic HN for fusion activation. Dye transfer assays show that the G3A and G7A mutations decrease the energy required to activate F at a step in the fusion cascade preceding prehairpin intermediate formation and hemifusion. Conserved glycine residues in the FP of paramyxovirus F appear to have a primary role in regulating the activation of the metastable native form of F. Glycine residues in the FPs of other class I vFGPs may also regulate fusion activation. PMID:15564482

The structural basis of chicken, swine and bovine CD8αα dimers provides insight into the co-evolution with MHC I in endotherm species

PubMed Central

Liu, Yanjie; Li, Xin; Qi, Jianxun; Zhang, Nianzhi; Xia, Chun

2016-01-01

It is unclear how the pivotal molecules of the adaptive immune system (AIS) maintain their inherent characteristics and relationships with their co-receptors over the course of co-evolution. CD8α, a fundamental but simple AIS component with only one immunoglobulin variable (IgV) domain, is a good example with which to explore this question because it can fold correctly to form homodimers (CD8αα) and interact with peptide-MHC I (p/MHC I) with low sequence identities between different species. Hereby, we resolved the crystal structures of chicken, swine and bovine CD8αα. They are typical homodimers consisting of two symmetric IgV domains with distinct species specificities. The CD8αα structures indicated that a few highly conserved residues are important in CD8 dimerization and in interacting with p/MHC I. The dimerization of CD8αα mainly depends on the pivotal residues on the dimer interface; in particular, four aromatic residues provide many intermolecular forces and contact areas. Three residues on the surface of CD8α connecting cavities that formed most of the hydrogen bonds with p/MHC I were also completely conserved. Our data propose that a few key conserved residues are able to ensure the CD8α own structural characteristics despite the great sequence variation that occurs during evolution in endotherms. PMID:27122108

Evolutionary and biophysical relationships among the papillomavirus E2 proteins.

PubMed

Blakaj, Dukagjin M; Fernandez-Fuentes, Narcis; Chen, Zigui; Hegde, Rashmi; Fiser, Andras; Burk, Robert D; Brenowitz, Michael

2009-01-01

Infection by human papillomavirus (HPV) may result in clinical conditions ranging from benign warts to invasive cancer. The HPV E2 protein represses oncoprotein transcription and is required for viral replication. HPV E2 binds to palindromic DNA sequences of highly conserved four base pair sequences flanking an identical length variable 'spacer'. E2 proteins directly contact the conserved but not the spacer DNA. Variation in naturally occurring spacer sequences results in differential protein affinity that is dependent on their sensitivity to the spacer DNA's unique conformational and/or dynamic properties. This article explores the biophysical character of this core viral protein with the goal of identifying characteristics that associated with risk of virally caused malignancy. The amino acid sequence, 3d structure and electrostatic features of the E2 protein DNA binding domain are highly conserved; specific interactions with DNA binding sites have also been conserved. In contrast, the E2 protein's transactivation domain does not have extensive surfaces of highly conserved residues. Rather, regions of high conservation are localized to small surface patches. Implications to cancer biology are discussed.

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 lipocalin fold. This information has been used to assign LIR2 to lipocalins and to investigate its structure/function relationship. This study could be applied to other protein families with low pairwise similarity, such as the structurally related fatty acid binding proteins or avidins. PMID:18190694

The complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface.

PubMed

Lorieau, Justin L; Louis, John M; Bax, Ad

2010-06-22

All but five of the N-terminal 23 residues of the HA2 domain of the influenza virus glycoprotein hemagglutinin (HA) are strictly conserved across all 16 serotypes of HA genes. The structure and function of this HA2 fusion peptide (HAfp) continues to be the focus of extensive biophysical, computational, and functional analysis, but most of these analyses are of peptides that do not include the strictly conserved residues Trp(21)-Tyr(22)-Gly(23). The heteronuclear triple resonance NMR study reported here of full length HAfp of sero subtype H1, solubilized in dodecylphosphatidyl choline, reveals a remarkably tight helical hairpin structure, with its N-terminal alpha-helix (Gly(1)-Gly(12)) packed tightly against its second alpha-helix (Trp(14)-Gly(23)), with six of the seven conserved Gly residues at the interhelical interface. The seventh conserved Gly residue in position 13 adopts a positive angle, enabling the hairpin turn that links the two helices. The structure is stabilized by multiple interhelical C(alpha)H to C=O hydrogen bonds, characterized by strong interhelical H(N)-H(alpha) and H(alpha)-H(alpha) NOE contacts. Many of the previously identified mutations that make HA2 nonfusogenic are also incompatible with the tight antiparallel hairpin arrangement of the HAfp helices.(15)N relaxation analysis indicates the structure to be highly ordered on the nanosecond time scale, and NOE analysis indicates HAfp is located at the water-lipid interface, with its hydrophobic surface facing the lipid environment, and the Gly-rich side of the helix-helix interface exposed to solvent.

The hypothetical protein Atu4866 from Agrobacterium tumefaciens adopts a streptavidin-like fold

PubMed Central

Ai, Xuanjun; Semesi, Anthony; Yee, Adelinda; Arrowsmith, Cheryl H.; Choy, Wing-Yiu; Li, Shawn S.C.

2008-01-01

Atu4866 is a 79-residue conserved hypothetical protein of unknown function from Agrobacterium tumefaciens. Protein sequence alignments show that it shares ≥60% sequence identity with 20 other hypothetical proteins of bacterial origin. However, the structures and functions of these proteins remain unknown so far. To gain insight into the function of this family of proteins, we have determined the structure of Atu4866 as a target of a structural genomics project using solution NMR spectroscopy. Our results reveal that Atu4866 adopts a streptavidin-like fold featuring a β-barrel/sandwich formed by eight antiparallel β-strands. Further structural analysis identified a continuous patch of conserved residues on the surface of Atu4866 that may constitute a potential ligand-binding site. PMID:18042676

The future of irrigation on the U.S. Great Plains

USDA-ARS?s Scientific Manuscript database

In the Great Plains, soil and water conservation is being achieved in both dryland and irrigated agricultural systems, and increasingly in combinations of these systems. Limiting tillage has increased the retention of crop residues on the surface and has reduced the evaporative loss of water, making...

On the relationship between residue structural environment and sequence conservation in proteins.

PubMed

Liu, Jen-Wei; Lin, Jau-Ji; Cheng, Chih-Wen; Lin, Yu-Feng; Hwang, Jenn-Kang; Huang, Tsun-Tsao

2017-09-01

Residues that are crucial to protein function or structure are usually evolutionarily conserved. To identify the important residues in protein, sequence conservation is estimated, and current methods rely upon the unbiased collection of homologous sequences. Surprisingly, our previous studies have shown that the sequence conservation is closely correlated with the weighted contact number (WCN), a measure of packing density for residue's structural environment, calculated only based on the C α positions of a protein structure. Moreover, studies have shown that sequence conservation is correlated with environment-related structural properties calculated based on different protein substructures, such as a protein's all atoms, backbone atoms, side-chain atoms, or side-chain centroid. To know whether the C α atomic positions are adequate to show the relationship between residue environment and sequence conservation or not, here we compared C α atoms with other substructures in their contributions to the sequence conservation. Our results show that C α positions are substantially equivalent to the other substructures in calculations of various measures of residue environment. As a result, the overlapping contributions between C α atoms and the other substructures are high, yielding similar structure-conservation relationship. Take the WCN as an example, the average overlapping contribution to sequence conservation is 87% between C α and all-atom substructures. These results indicate that only C α atoms of a protein structure could reflect sequence conservation at the residue level. © 2017 Wiley Periodicals, Inc.

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

PubMed Central

Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

2016-01-01

Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

PubMed

Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

2015-07-01

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

Structure-function Analysis of Receptor-binding in Adeno-Associated Virus Serotype 6 (AAV-6)

PubMed Central

Xie, Qing; Lerch, Thomas F.; Meyer, Nancy L.; Chapman, Michael S.

2011-01-01

Crystal structures of the AAV-6 capsid at 3 Å reveal a subunit fold homologous to other parvoviruses with greatest differences in two external loops. The electrostatic potential suggests that receptor-attachment is mediated by four residues: Arg576, Lys493, Lys459 and Lys531, defining a positively charged region curving up from the valley between adjacent spikes. It overlaps only partially with the receptor-binding site of AAV-2, and the residues endowing the electrostatic character are not homologous. Mutational substitution of each residue decreases heparin affinity, particularly Lys531 and Lys459. Neither is conserved among heparin-binding serotypes, indicating that diverse modes of receptor attachment have been selected in different serotypes. Surface topology and charge are also distinct at the shoulder of the spike, where linear epitopes for AAV-2’s neutralizing monoclonal antibody A20 come together. Evolutionarily, selection of changed side-chain charge may have offered a conservative means to evade immune neutralization while preserving other essential functionality. PMID:21917284

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

NASA Astrophysics Data System (ADS)

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

2016-02-01

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.

Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins*

PubMed Central

Meitzler, Jennifer L.; Hinde, Sara; Bánfi, Botond; Nauseef, William M.; Ortiz de Montellano, Paul R.

2013-01-01

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1. PMID:23362256

Gonadotropin-Releasing Hormone (GnRH) Receptor Structure and GnRH Binding

PubMed Central

Flanagan, Colleen A.; Manilall, Ashmeetha

2017-01-01

Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp3 and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on “conformationally constrained” GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function. PMID:29123501

Relationships between residue Voronoi volume and sequence conservation in proteins.

PubMed

Liu, Jen-Wei; Cheng, Chih-Wen; Lin, Yu-Feng; Chen, Shao-Yu; Hwang, Jenn-Kang; Yen, Shih-Chung

2018-02-01

Functional and biophysical constraints can cause different levels of sequence conservation in proteins. Previously, structural properties, e.g., relative solvent accessibility (RSA) and packing density of the weighted contact number (WCN), have been found to be related to protein sequence conservation (CS). The Voronoi volume has recently been recognized as a new structural property of the local protein structural environment reflecting CS. However, for surface residues, it is sensitive to water molecules surrounding the protein structure. Herein, we present a simple structural determinant termed the relative space of Voronoi volume (RSV); it uses the Voronoi volume and the van der Waals volume of particular residues to quantify the local structural environment. RSV (range, 0-1) is defined as (Voronoi volume-van der Waals volume)/Voronoi volume of the target residue. The concept of RSV describes the extent of available space for every protein residue. RSV and Voronoi profiles with and without water molecules (RSVw, RSV, VOw, and VO) were compared for 554 non-homologous proteins. RSV (without water) showed better Pearson's correlations with CS than did RSVw, VO, or VOw values. The mean correlation coefficient between RSV and CS was 0.51, which is comparable to the correlation between RSA and CS (0.49) and that between WCN and CS (0.56). RSV is a robust structural descriptor with and without water molecules and can quantitatively reflect evolutionary information in a single protein structure. Therefore, it may represent a practical structural determinant to study protein sequence, structure, and function relationships. Copyright © 2017 Elsevier B.V. All rights reserved.

A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation.

PubMed

Seal, R P; Amara, S G

1998-12-01

To investigate the structural determinants underlying transport by the glutamate transporter EAAT1, we mutated each of 24 highly conserved residues (P392 to Q415) to cysteine. A majority of these substituted cysteines react with the sulfhydryl-modifying reagent MTSEA, suggesting that they reside in an aqueous environment. The impermeant reagents MTSES and MTSET react with residues at each end of the domain (A395C and A414C), supporting a model that places these residues near the extracellular surface. Substrates and inhibitors block the reaction between MTS derivatives and A395C, and the cosubstrate, sodium, slows reaction of MTSEA with Y405C and E406C. From these results, we propose that this domain forms a reentrant membrane loop at the cell surface and may comprise part of the translocation pore for substrates and cotransported ions.

Evidence of a potential receptor-binding site on the Nipah virus G protein (NiV-G): identification of globular head residues with a role in fusion promotion and their localization on an NiV-G structural model.

PubMed

Guillaume, Vanessa; Aslan, Hamide; Ainouze, Michelle; Guerbois, Mathilde; Wild, T Fabian; Buckland, Robin; Langedijk, Johannes P M

2006-08-01

As a preliminary to the localization of the receptor-binding site(s) on the Nipah virus (NiV) glycoprotein (NiV-G), we have undertaken the identification of NiV-G residues that play a role in fusion promotion. To achieve this, we have used two strategies. First, as NiV and Hendra virus (HeV) share a common receptor and their cellular tropism is similar, we hypothesized that residues functioning in receptor attachment could be conserved between their respective G proteins. Our initial strategy was to target charged residues (which can be expected to be at the surface of the protein) conserved between the NiV-G and HeV-G globular heads. Second, we generated NiV variants that escaped neutralization by anti-NiV-G monoclonal antibodies (MAbs) that neutralize NiV both in vitro and in vivo, likely by blocking receptor attachment. The sequencing of such "escape mutants" identified NiV-G residues present in the epitopes to which the neutralizing MAbs are directed. Residues identified via these two strategies whose mutation had an effect on fusion promotion were localized on a new structural model for the NiV-G protein. Our results suggest that seven NiV-G residues, including one (E533) that was identified using both strategies, form a contiguous site on the top of the globular head that is implicated in ephrinB2 binding. This site commences near the shallow depression in the center of the top surface of the globular head and extends to the rim of the barrel-like structure on the top loops of beta-sheet 5. The topology of this site is strikingly similar to that proposed to form the SLAM receptor site on another paramyxovirus attachment protein, that of the measles virus hemagglutinin.

Evidence of a Potential Receptor-Binding Site on the Nipah Virus G Protein (NiV-G): Identification of Globular Head Residues with a Role in Fusion Promotion and Their Localization on an NiV-G Structural Model

PubMed Central

Guillaume, Vanessa; Aslan, Hamide; Ainouze, Michelle; Guerbois, Mathilde; Fabian Wild, T.; Buckland, Robin; Langedijk, Johannes P. M.

2006-01-01

As a preliminary to the localization of the receptor-binding site(s) on the Nipah virus (NiV) glycoprotein (NiV-G), we have undertaken the identification of NiV-G residues that play a role in fusion promotion. To achieve this, we have used two strategies. First, as NiV and Hendra virus (HeV) share a common receptor and their cellular tropism is similar, we hypothesized that residues functioning in receptor attachment could be conserved between their respective G proteins. Our initial strategy was to target charged residues (which can be expected to be at the surface of the protein) conserved between the NiV-G and HeV-G globular heads. Second, we generated NiV variants that escaped neutralization by anti-NiV-G monoclonal antibodies (MAbs) that neutralize NiV both in vitro and in vivo, likely by blocking receptor attachment. The sequencing of such “escape mutants” identified NiV-G residues present in the epitopes to which the neutralizing MAbs are directed. Residues identified via these two strategies whose mutation had an effect on fusion promotion were localized on a new structural model for the NiV-G protein. Our results suggest that seven NiV-G residues, including one (E533) that was identified using both strategies, form a contiguous site on the top of the globular head that is implicated in ephrinB2 binding. This site commences near the shallow depression in the center of the top surface of the globular head and extends to the rim of the barrel-like structure on the top loops of β-sheet 5. The topology of this site is strikingly similar to that proposed to form the SLAM receptor site on another paramyxovirus attachment protein, that of the measles virus hemagglutinin. PMID:16840334

Effects of Tillage, Cultivar and Fungicide on Phomopsis Longicolla and Cercospora Kukuchii in Soybean

USDA-ARS?s Scientific Manuscript database

It has become a standard practice for farmers to use a no-till production system where crop residue are left on the soil surface rather than incorporated into the soil. This practice helps reduce soil erosion, conserve energy, increase soil moisture, and reduce erosion. However, many plant pathogens...

Effect of tillage, cultivar and fungicide on Phomopsis longicolla and Cercospora kukuchii in soybean

USDA-ARS?s Scientific Manuscript database

It has become a standard practice for farmers to use a no-till production system where crop residue are left on the soil surface rather than incorporated into the soil. This practice helps reduce soil erosion, conserve energy, increase soil moisture, and reduce erosion. However, many plant pathogens...

Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure.

PubMed

Lakshmi, Balasubramanian; Sinduja, Chandrasekaran; Archunan, Govind; Srinivasan, Narayanaswamy

2014-06-01

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety-six of these alignment positions correspond to conserved Gly residue with (φ, ψ) values allowed for non-glycyl residues. Reasons for this observation were investigated by in-silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C(β) atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (φ, ψ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (φ, ψ) values which are disallowed for Ala. In-silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C(β) atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins. © 2014 The Protein Society.

A strategy for detecting the conservation of folding-nucleus residues in protein superfamilies.

PubMed

Michnick, S W; Shakhnovich, E

1998-01-01

Nucleation-growth theory predicts that fast-folding peptide sequences fold to their native structure via structures in a transition-state ensemble that share a small number of native contacts (the folding nucleus). Experimental and theoretical studies of proteins suggest that residues participating in folding nuclei are conserved among homologs. We attempted to determine if this is true in proteins with highly diverged sequences but identical folds (superfamilies). We describe a strategy based on comparisons of residue conservation in natural superfamily sequences with simulated sequences (generated with a Monte-Carlo sequence design strategy) for the same proteins. The basic assumptions of the strategy were that natural sequences will conserve residues needed for folding and stability plus function, the simulated sequences contain no functional conservation, and nucleus residues make native contacts with each other. Based on these assumptions, we identified seven potential nucleus residues in ubiquitin superfamily members. Non-nucleus conserved residues were also identified; these are proposed to be involved in stabilizing native interactions. We found that all superfamily members conserved the same potential nucleus residue positions, except those for which the structural topology is significantly different. Our results suggest that the conservation of the nucleus of a specific fold can be predicted by comparing designed simulated sequences with natural highly diverged sequences that fold to the same structure. We suggest that such a strategy could be used to help plan protein folding and design experiments, to identify new superfamily members, and to subdivide superfamilies further into classes having a similar folding mechanism.

Extrapolating effects of conservation tillage on yield, soil moisture and dry spell mitigation using simulation modelling

NASA Astrophysics Data System (ADS)

Mkoga, Z. J.; Tumbo, S. D.; Kihupi, N.; Semoka, J.

There is big effort to disseminate conservation tillage practices in Tanzania. Despite wide spread field demonstrations there has been some field experiments meant to assess and verify suitability of the tillage options in local areas. Much of the experiments are short lived and thus long term effects of the tillage options are unknown. Experiments to study long term effects of the tillage options are lacking because they are expensive and cannot be easily managed. Crop simulation models have the ability to use long term weather data and the local soil parameters to assess long term effects of the tillage practices. The Agricultural Production Systems Simulator (APSIM) crop simulation model; was used to simulate long term production series of soil moisture and grain yield based on the soil and weather conditions in Mkoji sub-catchment of the great Ruaha river basin in Tanzania. A 24 year simulated maize yield series based on conventional tillage with ox-plough, without surface crop residues (CT) treatment was compared with similar yield series based on conservation tillage (ox-ripping, with surface crop residues (RR)). Results showed that predicted yield averages were significantly higher in conservation tillage than in conventional tillage ( P < 0.001). Long term analysis, using APSIM simulation model, showed that average soil moisture in the conservation tillage was significantly higher ( P < 0.05) (about 0.29 mm/mm) than in conventional tillage (0.22 mm/mm) treatment during the seasons which received rainfall between 468 and 770 mm. Similarly the conservation tillage treatment recorded significantly higher yields (4.4 t/ha) ( P < 0.01) than the conventional tillage (3.6 t/ha) treatment in the same range of seasonal rainfall. On the other hand there was no significant difference in soil moisture for the seasons which received rainfall above 770 mm. In these seasons grain yield in conservation tillage treatment was significantly lower (3.1 kg/ha) than in the conventional tillage treatment (4.8 kg/ha) ( P < 0.05). Results also indicated a probability of 0.5 of getting higher yield in conservation than in conventional tillage practice. The conservation tillage treatment had the ability to even-out the acute and long intra-seasonal dry spells. For example a 36-days agricultural dry spell which occurred between 85th and 130th day after planting in the 1989/1990 season (in the CT treatment) was mitigated to zero days in the RR treatment by maintaining soil moisture above the critical point. Critical soil moisture for maize was measured at 0.55 of maximum soil moisture that can be depleted crop (0.55 D). It is concluded that conservation tillage practice where ripping and surface crop residues is used is much more effective in mitigating dry spells and increase productivity in a seasonal rainfall range of between 460 and 770 mm. It is recommended that farmers in the area adopt that type of conservation tillage because rainfall was in this range (460-770 mm) in 12 out of the past 24 years, indicating possibility of yield losses once in every 2 years.

Functional Sites Induce Long-Range Evolutionary Constraints in Enzymes

PubMed Central

Jack, Benjamin R.; Meyer, Austin G.; Echave, Julian; Wilke, Claus O.

2016-01-01

Functional residues in proteins tend to be highly conserved over evolutionary time. However, to what extent functional sites impose evolutionary constraints on nearby or even more distant residues is not known. Here, we report pervasive conservation gradients toward catalytic residues in a dataset of 524 distinct enzymes: evolutionary conservation decreases approximately linearly with increasing distance to the nearest catalytic residue in the protein structure. This trend encompasses, on average, 80% of the residues in any enzyme, and it is independent of known structural constraints on protein evolution such as residue packing or solvent accessibility. Further, the trend exists in both monomeric and multimeric enzymes and irrespective of enzyme size and/or location of the active site in the enzyme structure. By contrast, sites in protein–protein interfaces, unlike catalytic residues, are only weakly conserved and induce only minor rate gradients. In aggregate, these observations show that functional sites, and in particular catalytic residues, induce long-range evolutionary constraints in enzymes. PMID:27138088

STING Millennium: a web-based suite of programs for comprehensive and simultaneous analysis of protein structure and sequence

PubMed Central

Neshich, Goran; Togawa, Roberto C.; Mancini, Adauto L.; Kuser, Paula R.; Yamagishi, Michel E. B.; Pappas, Georgios; Torres, Wellington V.; Campos, Tharsis Fonseca e; Ferreira, Leonardo L.; Luna, Fabio M.; Oliveira, Adilton G.; Miura, Ronald T.; Inoue, Marcus K.; Horita, Luiz G.; de Souza, Dimas F.; Dominiquini, Fabiana; Álvaro, Alexandre; Lima, Cleber S.; Ogawa, Fabio O.; Gomes, Gabriel B.; Palandrani, Juliana F.; dos Santos, Gabriela F.; de Freitas, Esther M.; Mattiuz, Amanda R.; Costa, Ivan C.; de Almeida, Celso L.; Souza, Savio; Baudet, Christian; Higa, Roberto H.

2003-01-01

STING Millennium Suite (SMS) is a new web-based suite of programs and databases providing visualization and a complex analysis of molecular sequence and structure for the data deposited at the Protein Data Bank (PDB). SMS operates with a collection of both publicly available data (PDB, HSSP, Prosite) and its own data (contacts, interface contacts, surface accessibility). Biologists find SMS useful because it provides a variety of algorithms and validated data, wrapped-up in a user friendly web interface. Using SMS it is now possible to analyze sequence to structure relationships, the quality of the structure, nature and volume of atomic contacts of intra and inter chain type, relative conservation of amino acids at the specific sequence position based on multiple sequence alignment, indications of folding essential residue (FER) based on the relationship of the residue conservation to the intra-chain contacts and Cα–Cα and Cβ–Cβ distance geometry. Specific emphasis in SMS is given to interface forming residues (IFR)—amino acids that define the interactive portion of the protein surfaces. SMS may simultaneously display and analyze previously superimposed structures. PDB updates trigger SMS updates in a synchronized fashion. SMS is freely accessible for public data at http://www.cbi.cnptia.embrapa.br, http://mirrors.rcsb.org/SMS and http://trantor.bioc.columbia.edu/SMS. PMID:12824333

Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif.

PubMed

Rosenbaum, Sabrina; Kreft, Sandra; Etich, Julia; Frie, Christian; Stermann, Jacek; Grskovic, Ivan; Frey, Benjamin; Mielenz, Dirk; Pöschl, Ernst; Gaipl, Udo; Paulsson, Mats; Brachvogel, Bent

2011-02-18

Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.

A conserved motif in the linker domain of STAT1 transcription factor is required for both recognition and release from high-affinity DNA-binding sites.

PubMed

Hüntelmann, Bettina; Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas

2014-01-01

Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.

Weak conservation of structural features in the interfaces of homologous transient protein–protein complexes

PubMed Central

Sudha, Govindarajan; Singh, Prashant; Swapna, Lakshmipuram S; Srinivasan, Narayanaswamy

2015-01-01

Residue types at the interface of protein–protein complexes (PPCs) are known to be reasonably well conserved. However, we show, using a dataset of known 3-D structures of homologous transient PPCs, that the 3-D location of interfacial residues and their interaction patterns are only moderately and poorly conserved, respectively. Another surprising observation is that a residue at the interface that is conserved is not necessarily in the interface in the homolog. Such differences in homologous complexes are manifested by substitution of the residues that are spatially proximal to the conserved residue and structural differences at the interfaces as well as differences in spatial orientations of the interacting proteins. Conservation of interface location and the interaction pattern at the core of the interfaces is higher than at the periphery of the interface patch. Extents of variability of various structural features reported here for homologous transient PPCs are higher than the variation in homologous permanent homomers. Our findings suggest that straightforward extrapolation of interfacial nature and inter-residue interaction patterns from template to target could lead to serious errors in the modeled complex structure. Understanding the evolution of interfaces provides insights to improve comparative modeling of PPC structures. PMID:26311309

A conserved WW domain-like motif regulates invariant chain-dependent cell-surface transport of the NKG2D ligand ULBP2.

PubMed

Uhlenbrock, Franziska; van Andel, Esther; Andresen, Lars; Skov, Søren

2015-08-01

Malignant cells expressing NKG2D ligands on their cell surface can be directly sensed and killed by NKG2D-bearing lymphocytes. To ensure this immune recognition, accumulating evidence suggests that NKG2D ligands are trafficed via alternative pathways to the cell surface. We have previously shown that the NKG2D ligand ULBP2 traffics over an invariant chain (Ii)-dependent pathway to the cell surface. This study set out to elucidate how Ii regulates ULBP2 cell-surface transport: We discovered conserved tryptophan (Trp) residues in the primary protein sequence of ULBP1-6 but not in the related MICA/B. Substitution of Trp to alanine resulted in cell-surface inhibition of ULBP2 in different cancer cell lines. Moreover, the mutated ULBP2 constructs were retained and not degraded inside the cell, indicating a crucial role of this conserved Trp-motif in trafficking. Finally, overexpression of Ii increased surface expression of wt ULBP2 while Trp-mutants could not be expressed, proposing that this Trp-motif is required for an Ii-dependent cell-surface transport of ULBP2. Aberrant soluble ULBP2 is immunosuppressive. Thus, targeting a distinct protein module on the ULBP2 sequence could counteract this abnormal expression of ULBP2. Copyright © 2015 Elsevier Ltd. All rights reserved.

Zinc Binding and Dimerization of Streptococcus pyogenes Pyrogenic Exotoxin C Are Not Essential for T-Cell Stimulation

DTIC Science & Technology

2003-03-14

streptococcal superantigen binding to MHCII on the surface of cells (7–9), suggesting an essential role in both MHCII molecular recognition and TCR-mediated...extent, mutations of side chains found in a second conserved MHCII alpha-chain-binding site consisting of a hydrophobic surface loop decreased T-cell...fraction of dimer is present at T-cell stimulatory concentrations of Spe-C following mutation of the unpaired side chain of cys- teine at residue 27 to

p53 R175H hydrophobic patch and H-bond reorganization observed by MD simulation.

PubMed

Thayer, Kelly M; Quinn, Taylor R

2016-03-01

Molecular dynamics simulations probe the origins of aberrant functionality of R175H p53, which normally prevent tumorigenesis. This hotspot mutation exhibits loss of its essential zinc cofactor, aggregation, and activation of gain of function promoters, characteristics contributing to the loss of normal p53 activity. This study provided molecular level insight into the reorganization of the hydrogen bonding network and the formation of a hydrophobic patch on the surface of the protein. The hydrogen bonding network globally redistributes at the expense of the stability of the β-sandwich structure, and surface residues reorganize to expose a 250 Å(2) hydrophobic patch of residues covering approximately 2% of the solvent accessible surface. These changes could both stabilize the protein in the conformation exposing the patch to solvent to mediate the reported aggregation, and cause a destabilization in the area associated with DNA binding residues to affect the specificity. The development of the patch prior to loss of zinc indicates that stabilizing the patch quickly may prevent zinc loss. Considerations for rational design of small molecule therapeutics in light of the structural insight has been discussed and it suggest the positive ring around the hydrophobic patch and conserved residues may constitute a druggable site. © 2015 Wiley Periodicals, Inc.

Evolutionary Diversifaction of Aminopeptidase N in Lepidoptera by Conserved Clade-specific Amino Acid Residues

PubMed Central

Hughes, Austin L.

2015-01-01

Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family. PMID:24675701

The BID Domain of Type IV Secretion Substrates Forms a Conserved Four-Helix Bundle Topped with a Hook.

PubMed

Stanger, Frédéric V; de Beer, Tjaart A P; Dranow, David M; Schirmer, Tilman; Phan, Isabelle; Dehio, Christoph

2017-01-03

The BID (Bep intracellular delivery) domain functions as secretion signal in a subfamily of protein substrates of bacterial type IV secretion (T4S) systems. It mediates transfer of (1) relaxases and the attached DNA during bacterial conjugation, and (2) numerous Bartonella effector proteins (Beps) during protein transfer into host cells infected by pathogenic Bartonella species. Furthermore, BID domains of Beps have often evolved secondary effector functions within host cells. Here, we provide crystal structures for three representative BID domains and describe a novel conserved fold characterized by a compact, antiparallel four-helix bundle topped with a hook. The conserved hydrophobic core provides a rigid scaffold to a surface that, despite a few conserved exposed residues and similarities in charge distribution, displays significant variability. We propose that the genuine function of BID domains as T4S signal may primarily depend on their rigid structure, while the plasticity of their surface may facilitate adaptation to secondary effector functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

PubMed

Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

2016-02-01

Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation. © 2015 Wiley Periodicals, Inc.

Quantification and mapping of surface residue cover and tillage practices for maize and soybean fields in south central Nebraska-USA using Landsat imagery

USDA-ARS?s Scientific Manuscript database

The area cultivated under conservation tillage practices such as no-till and minimal tillage has recently increased in south central Nebraska (NE). Consequently, changes in some of the impacts of cropping systems on soil such as enhancing soil and water quality, improving soil structures and infiltr...

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

DOE PAGES

Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.; ...

2014-12-31

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

Structure of the extracellular domain of matrix protein 2 of influenza A virus in complex with a protective monoclonal antibody.

PubMed

Cho, Ki Joon; Schepens, Bert; Seok, Jong Hyeon; Kim, Sella; Roose, Kenny; Lee, Ji-Hye; Gallardo, Rodrigo; Van Hamme, Evelien; Schymkowitz, Joost; Rousseau, Frederic; Fiers, Walter; Saelens, Xavier; Kim, Kyung Hyun

2015-04-01

The extracellular domain of influenza A virus matrix protein 2 (M2e) is conserved and is being evaluated as a quasiuniversal influenza A vaccine candidate. We describe the crystal structure at 1.6 Å resolution of M2e in complex with the Fab fragment of an M2e-specific monoclonal antibody that protects against influenza A virus challenge. This antibody binds M2 expressed on the surfaces of cells infected with influenza A virus. Five out of six complementary determining regions interact with M2e, and three highly conserved M2e residues are critical for this interaction. In this complex, M2e adopts a compact U-shaped conformation stabilized in the center by the highly conserved tryptophan residue in M2e. This is the first description of the three-dimensional structure of M2e. M2e of influenza A is under investigation as a universal influenza A vaccine, but its three-dimensional structure is unknown. We describe the structure of M2e stabilized with an M2e-specific monoclonal antibody that recognizes natural M2. We found that the conserved tryptophan is positioned in the center of the U-shaped structure of M2e and stabilizes its conformation. The structure also explains why previously reported in vivo escape viruses, selected with a similar monoclonal antibody, carried proline residue substitutions at position 10 in M2. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Analysis of evolutionary conservation patterns and their influence on identifying protein functional sites.

PubMed

Fang, Chun; Noguchi, Tamotsu; Yamana, Hayato

2014-10-01

Evolutionary conservation information included in position-specific scoring matrix (PSSM) has been widely adopted by sequence-based methods for identifying protein functional sites, because all functional sites, whether in ordered or disordered proteins, are found to be conserved at some extent. However, different functional sites have different conservation patterns, some of them are linear contextual, some of them are mingled with highly variable residues, and some others seem to be conserved independently. Every value in PSSMs is calculated independently of each other, without carrying the contextual information of residues in the sequence. Therefore, adopting the direct output of PSSM for prediction fails to consider the relationship between conservation patterns of residues and the distribution of conservation scores in PSSMs. In order to demonstrate the importance of combining PSSMs with the specific conservation patterns of functional sites for prediction, three different PSSM-based methods for identifying three kinds of functional sites have been analyzed. Results suggest that, different PSSM-based methods differ in their capability to identify different patterns of functional sites, and better combining PSSMs with the specific conservation patterns of residues would largely facilitate the prediction.

Quality assessment of protein model-structures using evolutionary conservation.

PubMed

Kalman, Matan; Ben-Tal, Nir

2010-05-15

Programs that evaluate the quality of a protein structural model are important both for validating the structure determination procedure and for guiding the model-building process. Such programs are based on properties of native structures that are generally not expected for faulty models. One such property, which is rarely used for automatic structure quality assessment, is the tendency for conserved residues to be located at the structural core and for variable residues to be located at the surface. We present ConQuass, a novel quality assessment program based on the consistency between the model structure and the protein's conservation pattern. We show that it can identify problematic structural models, and that the scores it assigns to the server models in CASP8 correlate with the similarity of the models to the native structure. We also show that when the conservation information is reliable, the method's performance is comparable and complementary to that of the other single-structure quality assessment methods that participated in CASP8 and that do not use additional structural information from homologs. A perl implementation of the method, as well as the various perl and R scripts used for the analysis are available at http://bental.tau.ac.il/ConQuass/. nirb@tauex.tau.ac.il Supplementary data are available at Bioinformatics online.

The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction.

PubMed

Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J

2015-03-01

Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. © 2015. Published by The Company of Biologists Ltd.

Rapid comparison of protein binding site surfaces with Property Encoded Shape Distributions (PESD)

PubMed Central

Das, Sourav; Kokardekar, Arshad

2009-01-01

Patterns in shape and property distributions on the surface of binding sites are often conserved across functional proteins without significant conservation of the underlying amino-acid residues. To explore similarities of these sites from the viewpoint of a ligand, a sequence and fold-independent method was created to rapidly and accurately compare binding sites of proteins represented by property-mapped triangulated Gauss-Connolly surfaces. Within this paradigm, signatures for each binding site surface are produced by calculating their property-encoded shape distributions (PESD), a measure of the probability that a particular property will be at a specific distance to another on the molecular surface. Similarity between the signatures can then be treated as a measure of similarity between binding sites. As postulated, the PESD method rapidly detected high levels of similarity in binding site surface characteristics even in cases where there was very low similarity at the sequence level. In a screening experiment involving each member of the PDBBind 2005 dataset as a query against the rest of the set, PESD was able to retrieve a binding site with identical E.C. (Enzyme Commission) numbers as the top match in 79.5% of cases. The ability of the method in detecting similarity in binding sites with low sequence conservations were compared with state-of-the-art binding site comparison methods. PMID:19919089

An evolutionary analysis identifies a conserved pentapeptide stretch containing the two essential lysine residues for rice L-myo-inositol 1-phosphate synthase catalytic activity

PubMed Central

Basak, Papri; Maitra-Majee, Susmita; Das, Jayanta Kumar; Mukherjee, Abhishek; Ghosh Dastidar, Shubhra; Pal Choudhury, Pabitra

2017-01-01

A molecular evolutionary analysis of a well conserved protein helps to determine the essential amino acids in the core catalytic region. Based on the chemical properties of amino acid residues, phylogenetic analysis of a total of 172 homologous sequences of a highly conserved enzyme, L-myo-inositol 1-phosphate synthase or MIPS from evolutionarily diverse organisms was performed. This study revealed the presence of six phylogenetically conserved blocks, out of which four embrace the catalytic core of the functional protein. Further, specific amino acid modifications targeting the lysine residues, known to be important for MIPS catalysis, were performed at the catalytic site of a MIPS from monocotyledonous model plant, Oryza sativa (OsMIPS1). Following this study, OsMIPS mutants with deletion or replacement of lysine residues in the conserved blocks were made. Based on the enzyme kinetics performed on the deletion/replacement mutants, phylogenetic and structural comparison with the already established crystal structures from non-plant sources, an evolutionarily conserved peptide stretch was identified at the active pocket which contains the two most important lysine residues essential for catalytic activity. PMID:28950028

Multi-Seasonal Nitrogen Recoveries from Crop Residue in Soil and Crop in a Temperate Agro-Ecosystem

PubMed Central

Hu, Guoqing; Liu, Xiao; He, Hongbo; Zhang, Wei; Xie, Hongtu; Wu, Yeye; Cui, Jiehua; Sun, Ci; Zhang, Xudong

2015-01-01

In conservation tillage systems, at least 30% of the soil surface was covered by crop residues which generally contain significant amounts of nitrogen (N). However, little is known about the multi-seasonal recoveries of the N derived from these crop residues in soil-crop systems, notably in northeastern China. In a temperate agro-ecosystem, 15N-labeled maize residue was applied to field surfaces in the 1st year (2009). From the 2nd to 4th year (2010-2012), one treatment halted the application of maize residue, whereas the soil in the second treatment was re-applied with unlabeled maize residue. Crop and soil samples were collected after each harvest, and their 15N enrichments were determined on an isotope ratio mass spectrometer to trace the allocation of N derived from the initially applied maize residue in the soil-crop systems. On average, 8.4% of the maize residue N was recovered in the soil-crop in the 1st year, and the vast majority (61.9%-91.9%) was recovered during subsequent years. Throughout the experiment, the cumulative recovery of the residue N in the crop increased gradually (18.2%-20.9%), but most of the residue N was retained in the soil, notably in the 0-10 cm soil layer. Compared to the single application, the sequential residue application significantly increased the recovery of the residue N in the soil profile (73.8% vs. 40.9%) and remarkably decreased the total and the initially applied residue derived mineral N along the soil profile. Our results suggested that the residue N was actively involved in N cycling, and its release and recovery in crop and soil profile were controlled by the decomposition process. Sequential residue application significantly enhanced the retention and stabilization of the initially applied residue N in the soil and retarded its translocation along the soil profile. PMID:26192436

Structure of Alcohol Oxidase from Pichia pastoris by Cryo-Electron Microscopy

PubMed Central

Vonck, Janet; Parcej, David N.; Mills, Deryck J.

2016-01-01

The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes. PMID:27458710

Networks of high mutual information define the structural proximity of catalytic sites: implications for catalytic residue identification.

PubMed

Marino Buslje, Cristina; Teppa, Elin; Di Doménico, Tomas; Delfino, José María; Nielsen, Morten

2010-11-04

Identification of catalytic residues (CR) is essential for the characterization of enzyme function. CR are, in general, conserved and located in the functional site of a protein in order to attain their function. However, many non-catalytic residues are highly conserved and not all CR are conserved throughout a given protein family making identification of CR a challenging task. Here, we put forward the hypothesis that CR carry a particular signature defined by networks of close proximity residues with high mutual information (MI), and that this signature can be applied to distinguish functional from other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA) database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL) conservation measurement was shown to significantly outperform both the Shannon entropy and maximal frequency measurements. Residues in the proximity of catalytic sites were shown to be rich in shared MI. A structural proximity MI average score (termed pMI) was demonstrated to be a strong predictor for CR, thus confirming the proposed hypothesis. A structural proximity conservation average score (termed pC) was also calculated and demonstrated to carry distinct information from pMI. A catalytic likeliness score (Cls), combining the KL, pC and pMI measures, was shown to lead to significantly improved prediction accuracy. At a specificity of 0.90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function places limitations on the diversification of the structural environment along the course of evolution.

Chemical characteristics of water in the surficial aquifer system, Broward County, Florida

USGS Publications Warehouse

Howie, Barbara

1987-01-01

Water quality data was collected in 1981 and 1982 during the drilling of test holes at 27 sites throughout Broward County, Florida. Determinations were made for the following physical properties and chemical constituents: pH, alkalinity, specific conductance, major ions, selected nutrients and dissolved iron, aluminum, and manganese. Determinations for the trace elements-arsenic, barium, cadmium, chromium, lead, zinc, selenium, and mercury-were made at 14 wells. Water in the surficial aquifer system between the coastal ridge and the conservation areas is potable and usually is a calcium bicarbonate type for the first 140 ft or more below land surface. Between depths of 140 and 230 ft, groundwater generally grades into a mixed-ion water type. In some areas, diluted seawater occurs beneath the mixed water zone. Dissolved iron concentrations between the coastal ridge and the conservation areas are variable but generally exceed 1,000 micrograms/L. Beneath the conservation areas and the western edge of Broward County, groundwater in the first 100 ft below land surface generally is either a calcium bicarbonate type or a mixed-ion type. At depths between 100 and 200 ft, diluted residual seawater occurs, except along the far western edge of the county. Residual seawater is least diluted in the north. Dissolved iron concentrations generally are between 300 and 1 ,000 micrograms/L but increase to the east of the conservation areas. Other findings of the investigation include: (1) groundwater in some areas west of the coastal ridge probably would be suitable for most domestic, agricultural, and industrial uses if it were treated for carbonate hardness; (2) groundwater in much of Broward County is chemically altered by natural softening and magnesium enrichment (natural softening increases to the west and is very pronounced beneath the far western edge of the county); and (3) there is evidence of mineralized water from the conservation areas mixing with groundwater east of the levees. (Lantz-PTT)

A conserved catalytic residue in the ubiquitin-conjugating enzyme family

PubMed Central

Wu, Pei-Ying; Hanlon, Mary; Eddins, Michael; Tsui, Colleen; Rogers, Richard S.; Jensen, Jane P.; Matunis, Michael J.; Weissman, Allan M.; Wolberger, Cynthia P.; Pickart, Cecile M.

2003-01-01

Ubiquitin (Ub) regulates diverse functions in eukaryotes through its attachment to other proteins. The defining step in this protein modification pathway is the attack of a substrate lysine residue on Ub bound through its C-terminus to the active site cysteine residue of a Ub-conjugating enzyme (E2) or certain Ub ligases (E3s). So far, these E2 and E3 cysteine residues are the only enzyme groups known to participate in the catalysis of conjugation. Here we show that a strictly conserved E2 asparagine residue is critical for catalysis of E2- and E2/RING E3-dependent isopeptide bond formation, but dispensable for upstream and downstream reactions of Ub thiol ester formation. In constrast, the strictly conserved histidine and proline residues immediately upstream of the asparagine are dispensable for catalysis of isopeptide bond formation. We propose that the conserved asparagine side chain stabilizes the oxyanion intermediate formed during lysine attack. The E2 asparagine is the first non-covalent catalytic group to be proposed in any Ub conjugation factor. PMID:14517261

Identical phosphatase mechanisms achieved through distinct modes of binding phosphoprotein substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pazy, Y.; Motaleb, M.A.; Guarnieri, M.T.

2010-04-05

Two-component signal transduction systems are widespread in prokaryotes and control numerous cellular processes. Extensive investigation of sensor kinase and response regulator proteins from many two-component systems has established conserved sequence, structural, and mechanistic features within each family. In contrast, the phosphatases which catalyze hydrolysis of the response regulator phosphoryl group to terminate signal transduction are poorly understood. Here we present structural and functional characterization of a representative of the CheC/CheX/FliY phosphatase family. The X-ray crystal structure of Borrelia burgdorferi CheX complexed with its CheY3 substrate and the phosphoryl analogue BeF{sub 3}{sup -} reveals a binding orientation between a response regulatormore » and an auxiliary protein different from that shared by every previously characterized example. The surface of CheY3 containing the phosphoryl group interacts directly with a long helix of CheX which bears the conserved (E - X{sub 2} - N) motif. Conserved CheX residues Glu96 and Asn99, separated by a single helical turn, insert into the CheY3 active site. Structural and functional data indicate that CheX Asn99 and CheY3 Thr81 orient a water molecule for hydrolytic attack. The catalytic residues of the CheX-CheY3 complex are virtually superimposable on those of the Escherichia coli CheZ phosphatase complexed with CheY, even though the active site helices of CheX and CheZ are oriented nearly perpendicular to one other. Thus, evolution has found two structural solutions to achieve the same catalytic mechanism through different helical spacing and side chain lengths of the conserved acid/amide residues in CheX and CheZ.« less

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ovchinnikov, Victor; Louveau, Joy E.; Barton, John P.

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutationsmore » increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.« less

Gyrokinetic theory of turbulent acceleration and momentum conservation in tokamak plasmas

NASA Astrophysics Data System (ADS)

Lu, WANG; Shuitao, PENG; P, H. DIAMOND

2018-07-01

Understanding the generation of intrinsic rotation in tokamak plasmas is crucial for future fusion reactors such as ITER. We proposed a new mechanism named turbulent acceleration for the origin of the intrinsic parallel rotation based on gyrokinetic theory. The turbulent acceleration acts as a local source or sink of parallel rotation, i.e., volume force, which is different from the divergence of residual stress, i.e., surface force. However, the order of magnitude of turbulent acceleration can be comparable to that of the divergence of residual stress for electrostatic ion temperature gradient (ITG) turbulence. A possible theoretical explanation for the experimental observation of electron cyclotron heating induced decrease of co-current rotation was also proposed via comparison between the turbulent acceleration driven by ITG turbulence and that driven by collisionless trapped electron mode turbulence. We also extended this theory to electromagnetic ITG turbulence and investigated the electromagnetic effects on intrinsic parallel rotation drive. Finally, we demonstrated that the presence of turbulent acceleration does not conflict with momentum conservation.

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

DOE PAGES

Ovchinnikov, Victor; Louveau, Joy E.; Barton, John P.; ...

2018-02-14

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutationsmore » increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.« less

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

PubMed Central

2018-01-01

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutations increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed. PMID:29442996

Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stenmark, Pål; Dong, Min; Dupuy, Jérôme

2011-11-02

Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantlymore » reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.« less

Crystal structure of the botulinum neurotoxin type G binding domain: insight into cell surface binding.

PubMed

Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R; Stevens, Raymond C

2010-04-16

Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-A X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent. Copyright (c) 2010. Published by Elsevier Ltd.

Local Geometry and Evolutionary Conservation of Protein Surfaces Reveal the Multiple Recognition Patches in Protein-Protein Interactions

PubMed Central

Laine, Elodie; Carbone, Alessandra

2015-01-01

Protein-protein interactions (PPIs) are essential to all biological processes and they represent increasingly important therapeutic targets. Here, we present a new method for accurately predicting protein-protein interfaces, understanding their properties, origins and binding to multiple partners. Contrary to machine learning approaches, our method combines in a rational and very straightforward way three sequence- and structure-based descriptors of protein residues: evolutionary conservation, physico-chemical properties and local geometry. The implemented strategy yields very precise predictions for a wide range of protein-protein interfaces and discriminates them from small-molecule binding sites. Beyond its predictive power, the approach permits to dissect interaction surfaces and unravel their complexity. We show how the analysis of the predicted patches can foster new strategies for PPIs modulation and interaction surface redesign. The approach is implemented in JET2, an automated tool based on the Joint Evolutionary Trees (JET) method for sequence-based protein interface prediction. JET2 is freely available at www.lcqb.upmc.fr/JET2. PMID:26690684

Solution structure of the core SMN–Gemin2 complex

PubMed Central

Sarachan, Kathryn L.; Valentine, Kathleen G.; Gupta, Kushol; Moorman, Veronica R.; Gledhill, John M.; Bernens, Matthew; Tommos, Cecilia; Wand, A. Joshua; Van Duyne, Gregory D.

2012-01-01

In humans, assembly of spliceosomal snRNPs (small nuclear ribonucleoproteins) begins in the cytoplasm where the multi-protein SMN (survival of motor neuron) complex mediates the formation of a seven-membered ring of Sm proteins on to a conserved site of the snRNA (small nuclear RNA). The SMN complex contains the SMN protein Gemin2 and several additional Gemins that participate in snRNP biosynthesis. SMN was first identified as the product of a gene found to be deleted or mutated in patients with the neurodegenerative disease SMA (spinal muscular atrophy), the leading genetic cause of infant mortality. In the present study, we report the solution structure of Gemin2 bound to the Gemin2-binding domain of SMN determined by NMR spectroscopy. This complex reveals the structure of Gemin2, how Gemin2 binds to SMN and the roles of conserved SMN residues near the binding interface. Surprisingly, several conserved SMN residues, including the sites of two SMA patient mutations, are not required for binding to Gemin2. Instead, they form a conserved SMN/Gemin2 surface that may be functionally important for snRNP assembly. The SMN–Gemin2 structure explains how Gemin2 is stabilized by SMN and establishes a framework for structure–function studies to investigate snRNP biogenesis as well as biological processes involving Gemin2 that do not involve snRNP assembly. PMID:22607171

Setup errors and effectiveness of Optical Laser 3D Surface imaging system (Sentinel) in postoperative radiotherapy of breast cancer.

PubMed

Wei, Xiaobo; Liu, Mengjiao; Ding, Yun; Li, Qilin; Cheng, Changhai; Zong, Xian; Yin, Wenming; Chen, Jie; Gu, Wendong

2018-05-08

Breast-conserving surgery (BCS) plus postoperative radiotherapy has become the standard treatment for early-stage breast cancer. The aim of this study was to compare the setup accuracy of optical surface imaging by the Sentinel system with cone-beam computerized tomography (CBCT) imaging currently used in our clinic for patients received BCS. Two optical surface scans were acquired before and immediately after couch movement correction. The correlation between the setup errors as determined by the initial optical surface scan and CBCT was analyzed. The deviation of the second optical surface scan from the reference planning CT was considered an estimate for the residual errors for the new method for patient setup correction. The consequences in terms for necessary planning target volume (PTV) margins for treatment sessions without setup correction applied. We analyzed 145 scans in 27 patients treated for early stage breast cancer. The setup errors of skin marker based patient alignment by optical surface scan and CBCT were correlated, and the residual setup errors as determined by the optical surface scan after couch movement correction were reduced. Optical surface imaging provides a convenient method for improving the setup accuracy for breast cancer patient without unnecessary imaging dose.

Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase

PubMed Central

Lucas, James E; Siegel, Justin B

2015-01-01

Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses. PMID:25752240

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function

PubMed Central

Simpson, Brent W.; Owens, Tristan W.; Orabella, Matthew J.; Davis, Rebecca M.; May, Janine M.; Trauger, Sunia A.

2016-01-01

ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. PMID:27795402

A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat

PubMed Central

2014-01-01

Background The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. Results Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. Conclusions Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription. PMID:24985203

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yue; Zhao, Yuting; Su, Minfei

Mammalian Golgi-associated plant pathogenesis-related protein 1 (GAPR-1) is a negative autophagy regulator that binds Beclin 1, a key component of the autophagosome nucleation complex. Beclin 1 residues 267–284 are required for binding GAPR-1. Here, sequence analyses, structural modeling, mutagenesis combined with pull-down assays, X-ray crystal structure determination and small-angle X-ray scattering were used to investigate the Beclin 1–GAPR-1 interaction. Five conserved residues line an equatorial GAPR-1 surface groove that is large enough to bind a peptide. A model of a peptide comprising Beclin 1 residues 267–284 docked onto GAPR-1, built using theCABS-dockserver, indicates that this peptide binds to this GAPR-1more » groove. Mutation of the five conserved residues lining this groove, H54A/E86A/G102K/H103A/N138G, abrogates Beclin 1 binding. The 1.27 Å resolution X-ray crystal structure of this pentad mutant GAPR-1 was determined. Comparison with the wild-type (WT) GAPR-1 structure shows that the equatorial groove of the pentad mutant is shallower and more positively charged, and therefore may not efficiently bind Beclin 1 residues 267–284, which include many hydrophobic residues. Both WT and pentad mutant GAPR-1 crystallize as dimers, and in each case the equatorial groove of one subunit is partially occluded by the other subunit, indicating that dimeric GAPR-1 is unlikely to bind Beclin 1. SAXS analysis of WT and pentad mutant GAPR-1 indicates that in solution the WT forms monomers, while the pentad mutant is primarily dimeric. Thus, changes in the structure of the equatorial groove combined with the improved dimerization of pentad mutant GAPR-1 are likely to abrogate binding to Beclin 1.« less

Cleaning painted surfaces: evaluation of leaching phenomenon induced by solvents applied for the removal of gel residues.

PubMed

Casoli, Antonella; Di Diego, Zaira; Isca, Clelia

2014-12-01

Cleaning is one of the most important, delicate, and at the same time controversial processes in the conservation treatment of paintings. Although a strict definition of cleaning would be the removal of dirt, grime, or other accretions (surface cleaning), in the conservation field, cleaning is used in the broader meaning to include thinning/removing altered or “unwanted layers” of materials without damaging or altering the physicochemical properties of the surfaces to be preserved. The cleaning of unvarnished paintings is one of the most critical issues that are currently discussed. Several studies exist regarding different cleaning tools, such as gels, soaps, enzymes, ionic liquids, and foams, as well as various dry methods and lasers, but only a few have been performed on the risk associated with the use of water and organic solvents for the cleaning treatments in relation to the original paint binder. The aim of the study is to verify analytically the behavior of water gelling agents during cleaning treatments and the interaction of the following elements: water or organic solvents applied for the removal of gel residues with the original lipid paint binder. For this purpose, the study was conducted on a fragment of canvas painting (sixteenth to seventeenth century) of Soprintendenza per i Beni Storici, Artistici ed Etnoantropologici del Friuli Venezia Giulia (Superintendence for the Historical, Artistic and Ethno-anthropological Heritage of Friuli Venezia Giulia), Udine by means of Fourier transform infrared spectroscopy, gas chromatography/mass spectrometry, and scanning electron microscopy.

Reconnaissance survey of sulfonamide, sulfonylurea, and imidazolinone herbicides in surface streams and groundwater of the Midwestern United States

USGS Publications Warehouse

Steinheimer, T.R.; Pfeiffer, R.L.; Scoggin, K.D.; Battaglin, W.A.

2000-01-01

The study objective was to conduct a small scale synoptic survey of representative water resources draining agricultural land for occurrence of several herbicide residues. These new classes of herbicides are commonly applied pre-emergence or post-emergence in conservation tillage systems to control grasses and broadleaf weeds in cropped and noncropped areas. Both surface water and groundwater samples were collected from 44 midwestern locations during the summer of 1997, and analyzed for herbicide residues of 15 sulfonylurea and imidazolinone chemicals, and one sulfonamide. Each site was sampled between mid-June and late-October with several stream sites sampled twice. The method, developed jointly by the chemical manufacturer's and the U.S. Environmental Protection Agency, provides a 100 ng/L limit of quantitation in surface water for all analytes. Analytes were detected and identity confirmed in surface water at six sites and in ground water at two sites. The most frequently detected herbicides were imazaquin, imazethapyr, and nicosulfuron. For field studies in which the source of surface and ground water associated with the farming system on the agricultural landscape is known, the sensitivity of the method can be improved with only minor modifications in detection criteria.

A Tyrosine Residue on the TSH Receptor Stabilizes Multimer Formation

PubMed Central

Latif, Rauf; Michalek, Krzysztof; Morshed, Syed Ahmed; Davies, Terry F.

2010-01-01

Background The thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction. Methodology/Principal Findings We studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation. Conclusions/Significance These data establish the TSH receptor ectodomain as one site of multimerization, independent of the transmembrane region, and that this interaction was primarily via a conserved tyrosine residue in LRR3. PMID:20195479

POST-OPERATIONAL TREATMENT OF RESIDUAL NA COOLLANT IN EBR-2 USING CARBONATION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sherman, S.; Knight, C.

2011-03-08

At the end of 2002, the Experimental Breeder Reactor Two (EBR-II) facility became a U.S. Resource Conservation and Recovery Act (RCRA) permitted site, and the RCRA permit1 compelled further treatment of the residual sodium in order to convert it into a less reactive chemical form and remove the by-products from the facility, so that a state of RCRA 'closure' for the facility may be achieved (42 U.S.C. 6901-6992k, 2002). In response to this regulatory driver, and in recognition of project budgetary and safety constraints, it was decided to treat the residual sodium in the EBR-II primary and secondary sodium systemsmore » using a process known as 'carbonation.' In early EBR-II post-operation documentation, this process is also called 'passivation.' In the carbonation process (Sherman and Henslee, 2005), the system containing residual sodium is flushed with humidified carbon dioxide (CO{sub 2}). The water vapor in the flush gas reacts with residual sodium to form sodium hydroxide (NaOH), and the CO{sub 2} in the flush gas reacts with the newly formed NaOH to make sodium bicarbonate (NaHCO{sub 3}). Hydrogen gas (H{sub 2}) is produced as a by-product. The chemical reactions occur at the exposed surface of the residual sodium. The NaHCO{sub 3} layer that forms is porous, and humidified carbon dioxide can penetrate the NaHCO{sub 3} layer to continue reacting residual sodium underneath. The rate of reaction is controlled by the thickness of the NaHCO{sub 3} surface layer, the moisture input rate, and the residual sodium exposed surface area. At the end of carbonation, approximately 780 liters of residual sodium in the EBR-II primary tank ({approx}70% of original inventory), and just under 190 liters of residual sodium in the EBR-II secondary sodium system ({approx}50% of original inventory), were converted into NaHCO{sub 3}. No bare surfaces of residual sodium remained after treatment, and all remaining residual sodium deposits are covered by a layer of NaHCO{sub 3}. From a safety standpoint, the inventory of residual sodium in these systems was greatly reduced by using the carbonation process. From a regulatory standpoint, the process was not able to achieve deactivation of all residual sodium, and other more aggressive measures will be needed if the remaining residual sodium must also be deactivated to meet the requirements of the existing environmental permit. This chapter provides a project history and technical summary of the carbonation of EBR-II residual sodium. Options for future treatment are also discussed.« less

Structure of the Ebola VP35 interferon inhibitory domain.

PubMed

Leung, Daisy W; Ginder, Nathaniel D; Fulton, D Bruce; Nix, Jay; Basler, Christopher F; Honzatko, Richard B; Amarasinghe, Gaya K

2009-01-13

Ebola viruses (EBOVs) cause rare but highly fatal outbreaks of viral hemorrhagic fever in humans, and approved treatments for these infections are currently lacking. The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a viral assembly factor, and an inhibitor of host interferon (IFN) production. Mutation of select basic residues within the C-terminal half of VP35 abrogates its dsRNA-binding activity, impairs VP35-mediated IFN antagonism, and attenuates EBOV growth in vitro and in vivo. Because VP35 contributes to viral escape from host innate immunity and is required for EBOV virulence, understanding the structural basis for VP35 dsRNA binding, which correlates with suppression of IFN activity, is of high importance. Here, we report the structure of the C-terminal VP35 IFN inhibitory domain (IID) solved to a resolution of 1.4 A and show that VP35 IID forms a unique fold. In the structure, we identify 2 basic residue clusters, one of which is important for dsRNA binding. The dsRNA binding cluster is centered on Arg-312, a highly conserved residue required for IFN inhibition. Mutation of residues within this cluster significantly changes the surface electrostatic potential and diminishes dsRNA binding activity. The high-resolution structure and the identification of the conserved dsRNA binding residue cluster provide opportunities for antiviral therapeutic design. Our results suggest a structure-based model for dsRNA-mediated innate immune antagonism by Ebola VP35 and other similarly constructed viral antagonists.

Crystal Structure of Neurotropism-Associated Variable Surface Protein 1 (VSP1) of Borrelia Turicatae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lawson,C.; Yung, B.; Barbour, A.

2006-01-01

Vsp surface lipoproteins are serotype-defining antigens of relapsing fever spirochetes that undergo multiphasic antigenic variation to allow bacterial persistence in spite of an immune response. Two isogenic serotypes of Borrelia turicatae strain Oz1 differ in their Vsp sequences and in disease manifestations in infected mice: Vsp1 is associated with the selection of a neurological niche, while Vsp2 is associated with blood and skin infection. We report here crystal structures of the Vsp1 dimer at 2.7 and 2.2 Angstroms. The structures confirm that relapsing fever Vsp proteins share a common helical fold with OspCs of Lyme disease-causing Borrelia. The fold featuresmore » an inner stem formed by highly conserved N and C termini and an outer 'dome' formed by the variable central residues. Both Vsp1 and OspC structures possess small water-filled cavities, or pockets, that are lined largely by variable residues and are thus highly variable in shape. These features appear to signify tolerance of the Vsp-OspC fold for imperfect packing of residues at its antigenic surface. Structural comparison of Vsp1 with a homology model for Vsp2 suggests that observed differences in disease manifestation may arise in part from distinct differences in electrostatic surface properties; additional predicted positively charged surface patches on Vsp2 compared to Vsp1 may be sufficient to explain the relative propensity of Vsp2 to bind to acidic glycosaminoglycans.« less

ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove.

PubMed

Antoniou, Antony N; Santos, Susana G; Campbell, Elaine C; Lynch, Sarah; Arosa, Fernando A; Powis, Simon J

2007-05-15

The oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101-164, and 203-259 in the peptide-binding and alpha3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly.

Identification of a conserved 8 aa insert in the PIP5K protein in the Saccharomycetaceae family of fungi and the molecular dynamics simulations and structural analysis to investigate its potential functional role.

PubMed

Khadka, Bijendra; Gupta, Radhey S

2017-08-01

Homologs of the phosphatidylinositol-4-phosphate-5-kinase (PIP5K), which controls a multitude of essential cellular functions, contain a 8 aa insert in a conserved region that is specific for the Saccharomycetaceae family of fungi. Using structures of human PIP4K proteins as templates, structural models were generated of the Saccharomyces cerevisiae and human PIP5K proteins. In the modeled S. cerevisiae PIP5K, the 8 aa insert forms a surface exposed loop, present on the same face of the protein as the activation loop of the kinase domain. Electrostatic potential analysis indicates that the residues from 8 aa conserved loop form a highly positively charged surface patch, which through electrostatic interaction with the anionic portions of phospholipid head groups, is expected to play a role in the membrane interaction of the yeast PIP5K. To unravel this prediction, molecular dynamics (MD) simulations were carried out to examine the binding interaction of PIP5K, either containing or lacking the conserved signature insert, with two different membrane lipid bilayers. The results from MD studies provide insights concerning the mechanistic of interaction of PIP5K with lipid bilayer, and support the contention that the identified 8 aa conserved insert in fungal PIP5K plays an important role in the binding of this protein with membrane surface. Proteins 2017; 85:1454-1467. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Expression and mutagenesis of the sea anemone toxin Av2 reveals key amino acid residues important for activity on voltage-gated sodium channels.

PubMed

Moran, Yehu; Cohen, Lior; Kahn, Roy; Karbat, Izhar; Gordon, Dalia; Gurevitz, Michael

2006-07-25

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels (Na(v)s) and compete with the structurally dissimilar scorpion alpha-toxins on binding to receptor site-3. Although these features provide two structurally different probes for studying receptor site-3 and channel fast inactivation, the bioactive surface of sea anemone toxins has not been fully resolved. We established an efficient expression system for Av2 (known as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (previously named A. sulcata), and mutagenized it throughout. Each toxin mutant was analyzed in toxicity and binding assays as well as by circular dichroism spectroscopy to discern the effects derived from structural perturbation from those related to bioactivity. Six residues were found to constitute the anti-insect bioactive surface of Av2 (Val-2, Leu-5, Asn-16, Leu-18, and Ile-41). Further analysis of nine Av2 mutants on the human heart channel Na(v)1.5 expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects and mammals practically coincide but differ from the bioactive surface of a structurally similar sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica. Hence, our results not only demonstrate clear differences in the bioactive surfaces of Av2 and scorpion alpha-toxins but also indicate that despite the general conservation in structure and importance of the Arg-14 loop and its flanking residues Gly-10 and Gly-20 for function, the surface of interaction between different sea anemone toxins and Na(v)s varies.

Structural basis for the Nanos-mediated recruitment of the CCR4-NOT complex and translational repression.

PubMed

Bhandari, Dipankar; Raisch, Tobias; Weichenrieder, Oliver; Jonas, Stefanie; Izaurralde, Elisa

2014-04-15

The RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4-NOT deadenylase complex. Here, we show that the three human Nanos paralogs (Nanos1-3) interact with the CNOT1 C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1-3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1-3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. Our findings provide the structural basis for the recruitment of the CCR4-NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4-NOT complex as the main effector complex for Nanos function.

Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins

PubMed Central

De Jaco, Antonella; Dubi, Noga; Camp, Shelley; Taylor, Palmer

2017-01-01

The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination. PMID:23035660

An inter-residue network model to identify mutational-constrained regions on the Ebola coat glycoprotein

PubMed Central

Quinlan, Devin S.; Raman, Rahul; Tharakaraman, Kannan; Subramanian, Vidya; del Hierro, Gabriella; Sasisekharan, Ram

2017-01-01

Recently, progress has been made in the development of vaccines and monoclonal antibody cocktails that target the Ebola coat glycoprotein (GP). Based on the mutation rates for Ebola virus given its natural sequence evolution, these treatment strategies are likely to impose additional selection pressure to drive acquisition of mutations in GP that escape neutralization. Given the high degree of sequence conservation among GP of Ebola viruses, it would be challenging to determine the propensity of acquiring mutations in response to vaccine or treatment with one or a cocktail of monoclonal antibodies. In this study, we analyzed the mutability of each residue using an approach that captures the structural constraints on mutability based on the extent of its inter-residue interaction network within the three-dimensional structure of the trimeric GP. This analysis showed two distinct clusters of highly networked residues along the GP1-GP2 interface, part of which overlapped with epitope surfaces of known neutralizing antibodies. This network approach also permitted us to identify additional residues in the network of the known hotspot residues of different anti-Ebola antibodies that would impact antibody-epitope interactions. PMID:28397835

Residue-level resolution of alphavirus envelope protein interactions in pH-dependent fusion.

PubMed

Zeng, Xiancheng; Mukhopadhyay, Suchetana; Brooks, Charles L

2015-02-17

Alphavirus envelope proteins, organized as trimers of E2-E1 heterodimers on the surface of the pathogenic alphavirus, mediate the low pH-triggered fusion of viral and endosomal membranes in human cells. The lack of specific treatment for alphaviral infections motivates our exploration of potential antiviral approaches by inhibiting one or more fusion steps in the common endocytic viral entry pathway. In this work, we performed constant pH molecular dynamics based on an atomic model of the alphavirus envelope with icosahedral symmetry. We have identified pH-sensitive residues that cause the largest shifts in thermodynamic driving forces under neutral and acidic pH conditions for various fusion steps. A series of conserved interdomain His residues is identified to be responsible for the pH-dependent conformational changes in the fusion process, and ligand binding sites in their vicinity are anticipated to be potential drug targets aimed at inhibiting viral infections.

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.

In silico identification of functional regions in proteins.

PubMed

Nimrod, Guy; Glaser, Fabian; Steinberg, David; Ben-Tal, Nir; Pupko, Tal

2005-06-01

In silico prediction of functional regions on protein surfaces, i.e. sites of interaction with DNA, ligands, substrates and other proteins, is of utmost importance in various applications in the emerging fields of proteomics and structural genomics. When a sufficient number of homologs is found, powerful prediction schemes can be based on the observation that evolutionarily conserved regions are often functionally important, typically, only the principal functionally important region of the protein is detected, while secondary functional regions with weaker conservation signals are overlooked. Moreover, it is challenging to unambiguously identify the boundaries of the functional regions. We present a new methodology, called PatchFinder, that automatically identifies patches of conserved residues that are located in close proximity to each other on the protein surface. PatchFinder is based on the following steps: (1) Assignment of conservation scores to each amino acid position on the protein surface. (2) Assignment of a score to each putative patch, based on its likelihood to be functionally important. The patch of maximum likelihood is considered to be the main functionally important region, and the search is continued for non-overlapping patches of secondary importance. We examined the accuracy of the method using the IGPS enzyme, the SH2 domain and a benchmark set of 112 proteins. These examples demonstrated that PatchFinder is capable of identifying both the main and secondary functional patches. The PatchFinder program is available at: http://ashtoret.tau.ac.il/~nimrodg/

Dissociation of Paramyxovirus Interferon Evasion Activities: Universal and Virus-Specific Requirements for Conserved V Protein Amino Acids in MDA5 Interference ▿

PubMed Central

Ramachandran, Aparna; Horvath, Curt M.

2010-01-01

The V protein of the paramyxovirus subfamily Paramyxovirinae is an important virulence factor that can interfere with host innate immunity by inactivating the cytosolic pathogen recognition receptor MDA5. This interference is a result of a protein-protein interaction between the highly conserved carboxyl-terminal domain of the V protein and the helicase domain of MDA5. The V protein C-terminal domain (CTD) is an evolutionarily conserved 49- to 68-amino-acid region that coordinates two zinc atoms per protein chain. Site-directed mutagenesis of conserved residues in the V protein CTD has revealed both universal and virus-specific requirements for zinc coordination in MDA5 engagement and has also identified other conserved residues as critical for MDA5 interaction and interference. Mutation of these residues produces V proteins that are specifically defective for MDA5 interference and not impaired in targeting STAT1 for proteasomal degradation via the VDC ubiquitin ligase complex. Results demonstrate that mutation of conserved charged residues in the V proteins of Nipah virus, measles virus, and mumps virus also abolishes MDA5 interaction. These findings clearly define molecular determinants for MDA5 inhibition by the paramyxovirus V proteins. PMID:20719949

A novel protein-protein interaction in the RES (REtention and Splicing) complex.

PubMed

Tripsianes, Konstantinos; Friberg, Anders; Barrandon, Charlotte; Brooks, Mark; van Tilbeurgh, Herman; Seraphin, Bertrand; Sattler, Michael

2014-10-10

The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp(232) in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gromowski, Gregory D.; Barrett, Alan D.T.

2007-09-30

The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity ofmore » each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity ({approx} 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant.« less

Hematopoietic cytokines: similarities and differences in the structures, with implications for receptor binding.

PubMed Central

Wlodawer, A.; Pavlovsky, A.; Gustchina, A.

1993-01-01

Crystal and NMR structures of helical cytokines--interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-2 (IL-2)--have been compared. Root mean square deviations in the C alpha coordinates for the conserved regions of the helices were 1-2 A between different cytokines, about twice the differences observed for independently determined crystal and solution structures of IL-4. Considerable similarity in amino acid sequence in the areas expected to interact with the receptors was detected, and the available mutagenesis data for these cytokines were correlated with structure conservation. Models of cytokine-receptor interactions were postulated for IL-4 based on its structure as well as on the published structure of human growth hormone interacting with its receptors (de Vos, A.M., Ultsch, M., & Kossiakoff, A.A., 1992, Science 255, 306-312). Patches of positively charged residues on the surfaces of helices C and D of IL-4 may be responsible for the interactions with the negatively charged residues found in the complementary parts of the IL-4 receptors. PMID:8401223

Comprehensively Surveying Structure and Function of RING Domains from Drosophila melanogaster

PubMed Central

Wu, Yuehao; Wan, Fusheng; Huang, Chunhong; Jie, Kemin

2011-01-01

Using a complete set of RING domains from Drosophila melanogaster, all the solved RING domains and cocrystal structures of RING-containing ubiquitin-ligases (RING-E3) and ubiquitin-conjugating enzyme (E2) pairs, we analyzed RING domains structures from their primary to quarternary structures. The results showed that: i) putative orthologs of RING domains between Drosophila melanogaster and the human largely occur (118/139, 84.9%); ii) of the 118 orthologous pairs from Drosophila melanogaster and the human, 117 pairs (117/118, 99.2%) were found to retain entirely uniform domain architectures, only Iap2/Diap2 experienced evolutionary expansion of domain architecture; iii) 4 evolutionary structurally conserved regions (SCRs) are responsible for homologous folding of RING domains at the superfamily level; iv) besides the conserved Cys/His chelating zinc ions, 6 equivalent residues (4 hydrophobic and 2 polar residues) in the SCRs possess good-consensus and conservation- these 4 SCRs function in the structural positioning of 6 equivalent residues as determinants for RING-E3 catalysis; v) members of these RING proteins located nucleus, multiple subcellular compartments, membrane protein and mitochondrion are respectively 42 (42/139, 30.2%), 71 (71/139, 51.1%), 22 (22/139, 15.8%) and 4 (4/139, 2.9%); vi) CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than other RING-E3s; vii) analyzing intermolecular interfaces of E2/RING-E3 complexes indicate that residues directly interacting with E2s are all from the SCRs in RING domains. Of the 6 residues, 2 hydrophobic ones contribute to constructing the conserved hydrophobic core, while the 2 hydrophobic and 2 polar residues directly participate in E2/RING-E3 interactions. Based on sequence and structural data, SCRs, conserved equivalent residues and features of intermolecular interfaces were extracted, highlighting the presence of a nucleus for RING domain fold and formation of catalytic core in which related residues and regions exhibit preferential evolutionary conservation. PMID:21912646

Molecular cloning and functional expression of bovine spleen ecto-NAD+ glycohydrolase: structural identity with human CD38.

PubMed Central

Augustin, A; Muller-Steffner, H; Schuber, F

2000-01-01

Bovine spleen ecto-NAD(+) glycohydrolase, an archetypal member of the mammalian membrane-associated NAD(P)(+) glycohydrolase enzyme family (EC 3.2.2.6), displays catalytic features similar to those of CD38, i.e. a protein originally described as a lymphocyte differentiation marker involved in the metabolism of cyclic ADP-ribose and signal transduction. Using amino acid sequence information obtained from NAD(+) glycohydrolase and from a truncated and hydrosoluble form of the enzyme (hNADase) purified to homogeneity, a full-length cDNA clone was obtained. The deduced sequence indicates a protein of 278 residues with a molecular mass of 31.5 kDa. It predicts that bovine ecto-NAD(+) glycohydrolase is a type II transmembrane protein, with a very short intracellular tail. The bulk of the enzyme, which is extracellular and contains two potential N-glycosylation sites, yields the fully catalytically active hNADase which is truncated by 71 residues. Transfection of HeLa cells with the full-length cDNA resulted in the expression of the expected NAD(+) glycohydrolase, ADP-ribosyl cyclase and GDP-ribosyl cyclase activities at the surface of the cells. The bovine enzyme, which is the first 'classical' NAD(P)(+) glycohydrolase whose structure has been established, presents a particularly high sequence identity with CD38, including the presence of 10 strictly conserved cysteine residues in the ectodomain and putative catalytic residues. However, it lacks two otherwise conserved cysteine residues near its C-terminus. Thus hNADase, the truncated protein of 207 amino acids, represents the smallest functional domain endowed with all the catalytic activities of CD38/NAD(+) glycohydrolases so far identified. Altogether, our data strongly suggest that the cloned bovine spleen ecto-NAD(+) glycohydrolase is the bovine equivalent of CD38. PMID:10600637

A Conserved Region between the Heptad Repeats of Paramyxovirus Fusion Proteins is Critical for Proper F Protein Folding†

PubMed Central

Gardner, Amanda E.; Martin, Kimberly L.; Dutch, Rebecca E.

2008-01-01

Paramyxoviruses are a diverse family which utilizes a fusion (F) protein to enter cells via fusion of the viral lipid bilayer with a target cell membrane. Although certain regions of F are known to play critical roles in membrane fusion, the function of much of the protein remains unclear. Sequence alignment of a set of paramyxovirus F proteins and analysis utilizing Block Maker identified a region of conserved amino acid sequence in a large domain between the heptad repeats of F1, designated CBF1. We employed site-directed mutagenesis to analyze the function of completely conserved residues of CBF1 in both the simian virus 5 (SV5) and Hendra virus F proteins. The majority of CBF1 point mutants were deficient in homotrimer formation, proteolytic processing, and transport to the cell surface. For some SV5 F mutants, proteolytic cleavage and surface expression could be restored by expression at 30°C, and varying levels of fusion promotion were observed at this temperature. In addition, the mutant SV5 F V402A displayed a hyperfusogenic phenotype at both 30°C and 37°C, indicating this mutation allows for efficient fusion with only an extremely small amount of cleaved, active protein. The recently published prefusogenic structure of PIV5/SV5 F [Yin, H.S., et al. (2006) Nature 439, 38–44] indicates that residues within and flanking CBF1 interact with the fusion peptide domain. Together, these data suggest that CBF1-fusion peptide interactions are critical for the initial folding of paramyxovirus F proteins from across this important viral family, and can also modulate subsequent membrane fusion promotion. PMID:17417875

The X-Ray Crystal Structure of the Keratin 1-Keratin 10 Helix 2B Heterodimer Reveals Molecular Surface Properties and Biochemical Insights into Human Skin Disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bunick, Christopher G.; Milstone, Leonard M.

Keratins 1 (K1) and 10 (K10) are the primary keratins expressed in differentiated epidermis. Mutations in K1/K10 are associated with human skin diseases. We determined the crystal structure of the complex between the distal (2B) helices of K1 and K10 to better understand how human keratin structure correlates with function. The 3.3 Å resolution structure confirms many features inferred by previous biochemical analyses, but adds unexpected insights. It demonstrates a parallel, coiled-coil heterodimer with a predominantly hydrophobic intermolecular interface; this heterodimer formed a higher order complex with a second K1-K10-2B heterodimer via a Cys401K10 disulfide link, although the bond anglemore » is unanticipated. The molecular surface analysis of K1-K10-2B identified several pockets, one adjacent to the disulfide linkage and conserved in K5-K14. The solvent accessible surface area of the K1-K10 structure is 20–25% hydrophobic. The 2B region contains mixed acidic and basic patches proximally (N-terminal), whereas it is largely acidic distally (C-terminal). Mapping of conserved and nonconserved residues between K1-K10 and K5-K14 onto the structure demonstrated the majority of unique residues align along the outer helical ridge. Finally, the structure permitted a fresh analysis of the deleterious effects caused by K1/K10 missense mutations found in patients with phenotypic skin disease.« less

The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi

2008-07-29

Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains onmore » the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.« less

Crystal Structure of a Fibroblast Growth Factor Homologous Factor (FHF) Defines a Conserved Surface on FHFs for Binding and Modulation of Voltage-gated Sodium Channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goetz, R.; Dover, K; Laezza, F

2009-01-01

Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav alpha subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel bindingmore » in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs.« less

Protein structure based prediction of catalytic residues.

PubMed

Fajardo, J Eduardo; Fiser, Andras

2013-02-22

Worldwide structural genomics projects continue to release new protein structures at an unprecedented pace, so far nearly 6000, but only about 60% of these proteins have any sort of functional annotation. We explored a range of features that can be used for the prediction of functional residues given a known three-dimensional structure. These features include various centrality measures of nodes in graphs of interacting residues: closeness, betweenness and page-rank centrality. We also analyzed the distance of functional amino acids to the general center of mass (GCM) of the structure, relative solvent accessibility (RSA), and the use of relative entropy as a measure of sequence conservation. From the selected features, neural networks were trained to identify catalytic residues. We found that using distance to the GCM together with amino acid type provide a good discriminant function, when combined independently with sequence conservation. Using an independent test set of 29 annotated protein structures, the method returned 411 of the initial 9262 residues as the most likely to be involved in function. The output 411 residues contain 70 of the annotated 111 catalytic residues. This represents an approximately 14-fold enrichment of catalytic residues on the entire input set (corresponding to a sensitivity of 63% and a precision of 17%), a performance competitive with that of other state-of-the-art methods. We found that several of the graph based measures utilize the same underlying feature of protein structures, which can be simply and more effectively captured with the distance to GCM definition. This also has the added the advantage of simplicity and easy implementation. Meanwhile sequence conservation remains by far the most influential feature in identifying functional residues. We also found that due the rapid changes in size and composition of sequence databases, conservation calculations must be recalibrated for specific reference databases.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

PubMed

Du, Yushen; Wu, Nicholas C; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting; Sun, Ren

2016-11-01

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available. Copyright © 2016 Du et al.

Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae.

PubMed

Stevenson, Andrew; Macdonald, Julie; Roberts, Mark

2003-03-20

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumoniae. Little is known about the mechanisms by which A. pleuropneumoniae colonises the respiratory tract. Fimbriae are common mediators of bacterial adherence to mucosal epithelia and have been observed on the surface of A. pleuropneumoniae cells. Here we report the identification and characterisation of the type 4 fimbrial structural gene (apfA) from A. pleuropneumoniae. In addition a number of open reading frames were identified in A. pleuropneumoniae that have significant homology to type 4 fimbrial biogenesis genes from other species, including a putative leader specific peptidase (apfD). A. pleuropneumoniae apfA codes for a predicted polypeptide of approximately 16kDa, removal of the leader sequence at the predicted cleavage site would yield a 14.5kDa polypeptide. The first 30 residues of the mature polypeptide are well conserved with other members of the group A type 4 fimbriae family. The signal sequence of ApfA is 13 amino acids in length and, unusually, the residue that precedes the cleavage site is alanine rather than glycine which is found in most other type 4 fimbriae. The C-terminus of ApfA possesses cysteine residues that are conserved in type 4 fimbriae of many species. In other type 4 fimbriae the distal C-terminal cysteines form a disulphide bond that produces a loop, which is important for the function of fimbriae and also comprises a major antigenic determinant. A motif within the predicted loop in ApfA was found to be highly conserved in type 4 fimbriae of other HAP organisms (Haemophilus, Actinobacillus, Pasteurella). The A. pleuropneumoniae type 4 fimbrial biogenesis genes showed the strongest homology to putative type 4 fimbrial genes of Haemophilus ducreyi. A. pleuropneumoniae apfA gene was shown to be present and highly conserved in different serotypes of A. pleuropneumoniae. Recombinant ApfA was produced and used to raise anti-ApfA antisera.

Identification, characterization, and functional analysis of Tube and Pelle homologs in the mud crab Scylla paramamosain.

PubMed

Li, Xin-Cang; Zhang, Xiao-Wen; Zhou, Jun-Fang; Ma, Hong-Yu; Liu, Zhi-Dong; Zhu, Lei; Yao, Xiao-Juan; Li, Lin-Gui; Fang, Wen-Hong

2013-01-01

Tube and Pelle are essential components in Drosophila Toll signaling pathway. In this study, we characterized a pair of crustacean homologs of Tube and Pelle in Scylla paramamosain, namely, SpTube and SpPelle, and analyzed their immune functions. The full-length cDNA of SpTube had 2052 bp with a 1578 bp open reading frame (ORF) encoding a protein with 525 aa. A death domain (DD) and a kinase domain were predicted in the deduced protein. The full-length cDNA of SpPelle had 3825 bp with a 3420 bp ORF encoding a protein with 1140 aa. The protein contained a DD and a kinase domain. Two conserved repeat motifs previously called Tube repeat motifs present only in insect Tube or Tube-like sequences were found between these two domains. Alignments and structure predictions demonstrated that SpTubeDD and SpPelleDD significantly differed in sequence and 3D structure. Similar to TubeDD, SpTubeDD contained three common conserved residues (R, K, and R) on one surface that may mediate SpMyD88 binding and two common residues (A and A) on the other surface that may contribute to Pelle binding. By contrast, SpPelleDD lacked similar conservative residues. SpTube, insect Tube-like kinases, and human IRAK4 were found to be RD kinases with an RD dipeptide in the kinase domain. SpPelle, Pelle, insect Pelle-like kinases, and human IRAK1 were found to be non-RD kinases lacking an RD dipeptide. Both SpTube and SpPelle were highly expressed in hemocytes, gills, and hepatopancreas. Upon challenge, SpTube and SpPele were significantly increased in hemocytes by Gram-negative or Gram-positive bacteria, whereas only SpPelle was elevated by White Spot Syndrome Virus. The pull-down assay showed that SpTube can bind to both SpMyD88 and SpPelle. These results suggest that SpTube, SpPelle, and SpMyD88 may form a trimeric complex involved in the immunity of mud crabs against both Gram-negative and Gram-positive bacteria.

Identification, Characterization, and Functional Analysis of Tube and Pelle Homologs in the Mud Crab Scylla paramamosain

PubMed Central

Zhou, Jun-Fang; Ma, Hong-Yu; Liu, Zhi-Dong; Zhu, Lei; Yao, Xiao-Juan; Li, Lin-Gui; Fang, Wen-Hong

2013-01-01

Tube and Pelle are essential components in Drosophila Toll signaling pathway. In this study, we characterized a pair of crustacean homologs of Tube and Pelle in Scylla paramamosain, namely, SpTube and SpPelle, and analyzed their immune functions. The full-length cDNA of SpTube had 2052 bp with a 1578 bp open reading frame (ORF) encoding a protein with 525 aa. A death domain (DD) and a kinase domain were predicted in the deduced protein. The full-length cDNA of SpPelle had 3825 bp with a 3420 bp ORF encoding a protein with 1140 aa. The protein contained a DD and a kinase domain. Two conserved repeat motifs previously called Tube repeat motifs present only in insect Tube or Tube-like sequences were found between these two domains. Alignments and structure predictions demonstrated that SpTubeDD and SpPelleDD significantly differed in sequence and 3D structure. Similar to TubeDD, SpTubeDD contained three common conserved residues (R, K, and R) on one surface that may mediate SpMyD88 binding and two common residues (A and A) on the other surface that may contribute to Pelle binding. By contrast, SpPelleDD lacked similar conservative residues. SpTube, insect Tube-like kinases, and human IRAK4 were found to be RD kinases with an RD dipeptide in the kinase domain. SpPelle, Pelle, insect Pelle-like kinases, and human IRAK1 were found to be non-RD kinases lacking an RD dipeptide. Both SpTube and SpPelle were highly expressed in hemocytes, gills, and hepatopancreas. Upon challenge, SpTube and SpPele were significantly increased in hemocytes by Gram-negative or Gram-positive bacteria, whereas only SpPelle was elevated by White Spot Syndrome Virus. The pull-down assay showed that SpTube can bind to both SpMyD88 and SpPelle. These results suggest that SpTube, SpPelle, and SpMyD88 may form a trimeric complex involved in the immunity of mud crabs against both Gram-negative and Gram-positive bacteria. PMID:24116143

Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1.

PubMed

Hannemann, Liya; Suppanz, Ida; Ba, Qiaorui; MacInnes, Katherine; Drepper, Friedel; Warscheid, Bettina; Koch, Hans-Georg

2016-01-20

YchF/Ola1 are unconventional members of the universally conserved GTPase family because they preferentially hydrolyze ATP rather than GTP. These ATPases have been associated with various cellular processes and pathologies, including DNA repair, tumorigenesis, and apoptosis. In particular, a possible role in regulating the oxidative stress response has been suggested for both bacterial and human YchF/Ola1. In this study, we analyzed how YchF responds to oxidative stress and how it potentially regulates the antioxidant response. Our data identify a redox-regulated monomer-dimer equilibrium of YchF as a key event in the functional cycle of YchF. Upon oxidative stress, the oxidation of a conserved and surface-exposed cysteine residue promotes YchF dimerization, which is accompanied by inhibition of the ATPase activity. No dimers were observed in a YchF mutant lacking this cysteine. In vitro, the YchF dimer is dissociated by thioredoxin 1 (TrxA) and this stimulates the ATPase activity. The physiological significance of the YchF-thioredoxin 1 interaction was demonstrated by in vivo cross-linking, which validated this interaction in living cells. This approach also revealed that both the ATPase domain and the helical domain of YchF are in contact with TrxA. YchF/Ola1 are the first redox-regulated members of the universally conserved GTPase family and are inactivated by oxidation of a conserved cysteine residue within the nucleotide-binding motif. Our data provide novel insights into the regulation of the so far ill-defined YchF/Ola1 family of proteins and stipulate their role as negative regulators of the oxidative stress response.

Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1

PubMed Central

Hannemann, Liya; Suppanz, Ida; Ba, Qiaorui; MacInnes, Katherine; Drepper, Friedel; Warscheid, Bettina

2016-01-01

Abstract Aims: YchF/Ola1 are unconventional members of the universally conserved GTPase family because they preferentially hydrolyze ATP rather than GTP. These ATPases have been associated with various cellular processes and pathologies, including DNA repair, tumorigenesis, and apoptosis. In particular, a possible role in regulating the oxidative stress response has been suggested for both bacterial and human YchF/Ola1. In this study, we analyzed how YchF responds to oxidative stress and how it potentially regulates the antioxidant response. Results: Our data identify a redox-regulated monomer–dimer equilibrium of YchF as a key event in the functional cycle of YchF. Upon oxidative stress, the oxidation of a conserved and surface-exposed cysteine residue promotes YchF dimerization, which is accompanied by inhibition of the ATPase activity. No dimers were observed in a YchF mutant lacking this cysteine. In vitro, the YchF dimer is dissociated by thioredoxin 1 (TrxA) and this stimulates the ATPase activity. The physiological significance of the YchF-thioredoxin 1 interaction was demonstrated by in vivo cross-linking, which validated this interaction in living cells. This approach also revealed that both the ATPase domain and the helical domain of YchF are in contact with TrxA. Innovation: YchF/Ola1 are the first redox-regulated members of the universally conserved GTPase family and are inactivated by oxidation of a conserved cysteine residue within the nucleotide-binding motif. Conclusion: Our data provide novel insights into the regulation of the so far ill-defined YchF/Ola1 family of proteins and stipulate their role as negative regulators of the oxidative stress response. Antioxid. Redox Signal. 24, 141–156. PMID:26160547

A residual-based shock capturing scheme for the continuous/discontinuous spectral element solution of the 2D shallow water equations

NASA Astrophysics Data System (ADS)

Marras, Simone; Kopera, Michal A.; Constantinescu, Emil M.; Suckale, Jenny; Giraldo, Francis X.

2018-04-01

The high-order numerical solution of the non-linear shallow water equations is susceptible to Gibbs oscillations in the proximity of strong gradients. In this paper, we tackle this issue by presenting a shock capturing model based on the numerical residual of the solution. Via numerical tests, we demonstrate that the model removes the spurious oscillations in the proximity of strong wave fronts while preserving their strength. Furthermore, for coarse grids, it prevents energy from building up at small wave-numbers. When applied to the continuity equation to stabilize the water surface, the addition of the shock capturing scheme does not affect mass conservation. We found that our model improves the continuous and discontinuous Galerkin solutions alike in the proximity of sharp fronts propagating on wet surfaces. In the presence of wet/dry interfaces, however, the model needs to be enhanced with the addition of an inundation scheme which, however, we do not address in this paper.

A Structural Biology and Protein Engineering Approach to the Development of Antidotes Against the Inhibition of Human Acetylcholinesterase by OP-based Nerve Agents

DTIC Science & Technology

2015-05-01

structures that we reported earlier (Kryger et al [2000] Acta Crystallogr D Biol Crystallogr 56:1385-1394) were of complexes with the snake venom...interactions between conserved residues in the loop connecting α13 to α14 and residues from helices α18’-α19’, and, conversely, between residues in the...residues in the 4-helix bundle, including Glu376, Thr383, Asp384, Trp385, Gln508, Gln527, Phe535 and Lys538 (hAChE numbering), are strictly conserved in

Conservation of artists' acrylic emulsion paints: XPS, NEXAFS and ATR-FTIR studies of wet cleaning methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willneff, E. A.; Ormsby, B. A.; Stevens, J. S.

Works of art prepared with acrylic emulsion paints became commercially available in the 1960s. It is increasingly necessary to undertake and optimise cleaning and preventative conservation treatments to ensure their longevity. Model artists' acrylic paint films covered with artificial soiling were thus prepared on a canvas support and exposed to a variety of wet cleaning treatments based on aqueous or hydrocarbon solvent systems. This included some with additives such as chelating agents and/or surfactants, and microemulsion systems made specifically for conservation practice. The impact of cleaning (soiling removal) on the paint film surface was examined visually and correlated with resultsmore » of attenuated total reflection Fourier transform infrared, XPS and near-edge X-ray absorption fine structure analyses – three spectroscopic techniques with increasing surface sensitivity ranging from approximately $-$ 1000, 10 and 5 nm, respectively. Visual analysis established the relative cleaning efficacy of the wet cleaning treatments in line with previous results. X-ray spectroscopy analysis provided significant additional findings, including evidence for (i) surfactant extraction following aqueous swabbing, (ii) modifications to pigment following cleaning and (iii) cleaning system residues.« less

Conservation of artists' acrylic emulsion paints: XPS, NEXAFS and ATR-FTIR studies of wet cleaning methods

DOE PAGES

Willneff, E. A.; Ormsby, B. A.; Stevens, J. S.; ...

2014-02-17

Works of art prepared with acrylic emulsion paints became commercially available in the 1960s. It is increasingly necessary to undertake and optimise cleaning and preventative conservation treatments to ensure their longevity. Model artists' acrylic paint films covered with artificial soiling were thus prepared on a canvas support and exposed to a variety of wet cleaning treatments based on aqueous or hydrocarbon solvent systems. This included some with additives such as chelating agents and/or surfactants, and microemulsion systems made specifically for conservation practice. The impact of cleaning (soiling removal) on the paint film surface was examined visually and correlated with resultsmore » of attenuated total reflection Fourier transform infrared, XPS and near-edge X-ray absorption fine structure analyses – three spectroscopic techniques with increasing surface sensitivity ranging from approximately $-$ 1000, 10 and 5 nm, respectively. Visual analysis established the relative cleaning efficacy of the wet cleaning treatments in line with previous results. X-ray spectroscopy analysis provided significant additional findings, including evidence for (i) surfactant extraction following aqueous swabbing, (ii) modifications to pigment following cleaning and (iii) cleaning system residues.« less

Structural Determinants of Sleeping Beauty Transposase Activity

PubMed Central

Abrusán, György; Yant, Stephen R; Szilágyi, András; Marsh, Joseph A; Mátés, Lajos; Izsvák, Zsuzsanna; Barabás, Orsolya; Ivics, Zoltán

2016-01-01

Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called “sectors”, which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold. PMID:27401040

Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation.

PubMed

Ng, Chai-Ann; Gravel, Andrée E; Perry, Matthew D; Arnold, Alexandre A; Marcotte, Isabelle; Vandenberg, Jamie I

2016-08-12

Slow deactivation of Kv11.1 channels is critical for its function in the heart. The S4-S5 linker, which joins the voltage sensor and pore domains, plays a critical role in this slow deactivation gating. Here, we use NMR spectroscopy to identify the membrane-bound surface of the S4S5 linker, and we show that two highly conserved tyrosine residues within the KCNH subfamily of channels are membrane-associated. Site-directed mutagenesis and electrophysiological analysis indicates that Tyr-542 interacts with both the pore domain and voltage sensor residues to stabilize activated conformations of the channel, whereas Tyr-545 contributes to the slow kinetics of deactivation by primarily stabilizing the transition state between the activated and closed states. Thus, the two tyrosine residues in the Kv11.1 S4S5 linker play critical but distinct roles in the slow deactivation phenotype, which is a hallmark of Kv11.1 channels. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeting cysteine residues of human immunodeficiency virus type 1 protease by reactive free radical species.

PubMed

Basu, A; Sehajpal, P K; Ogiste, J S; Lander, H M

1999-01-01

Nitric oxide (NO) is a naturally occurring free radical with many functions. The oxidized form of NO, the nitrosonium ion, reacts with the thiol group of cysteine residues resulting in their modification to S-nitrosothiols. The human immunodeficiency virus type 1 (HIV-1) protease (HIV-PR) has two cysteine residues that are conserved amongst different viral isolates found in patients with acquired immunodeficiency syndrome (AIDS). In an active dimer, these residues are located near the surface of the protease. We have found that treatment of HIV-PR with different NO congeners results in loss of its proteolytic activity and simultaneous formation of S-nitrosothiols. Sodium nitroprusside inhibited HIV-PR up to 70% and S-nitroso-N-acetylpenicillamine completely inhibited the protease within 5 min of treatment. The pattern of inhibition by NO donors is comparable to its inhibition by N-acetyl pepstatin. Using electrospray ionization-mass spectrometry, we identified the modification of HIV-PR by NO as that of S-nitrosation. Our findings point towards a possible role of NO in mediating resistance to HIV-1 infection.

Combining protein sequence, structure, and dynamics: A novel approach for functional evolution analysis of PAS domain superfamily.

PubMed

Dong, Zheng; Zhou, Hongyu; Tao, Peng

2018-02-01

PAS domains are widespread in archaea, bacteria, and eukaryota, and play important roles in various functions. In this study, we aim to explore functional evolutionary relationship among proteins in the PAS domain superfamily in view of the sequence-structure-dynamics-function relationship. We collected protein sequences and crystal structure data from RCSB Protein Data Bank of the PAS domain superfamily belonging to three biological functions (nucleotide binding, photoreceptor activity, and transferase activity). Protein sequences were aligned and then used to select sequence-conserved residues and build phylogenetic tree. Three-dimensional structure alignment was also applied to obtain structure-conserved residues. The protein dynamics were analyzed using elastic network model (ENM) and validated by molecular dynamics (MD) simulation. The result showed that the proteins with same function could be grouped by sequence similarity, and proteins in different functional groups displayed statistically significant difference in their vibrational patterns. Interestingly, in all three functional groups, conserved amino acid residues identified by sequence and structure conservation analysis generally have a lower fluctuation than other residues. In addition, the fluctuation of conserved residues in each biological function group was strongly correlated with the corresponding biological function. This research suggested a direct connection in which the protein sequences were related to various functions through structural dynamics. This is a new attempt to delineate functional evolution of proteins using the integrated information of sequence, structure, and dynamics. © 2017 The Protein Society.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruvinsky, Anatoly M., E-mail: anatoly.ruvinsky@astrazeneca.com; Center for Bioinformatics, The University of Kansas, Lawrence, Kansas 66047; Vakser, Ilya A.

Ferritin-like molecules show a remarkable combination of the evolutionary conserved activity of iron uptake and release that engage different pores in the conserved ferritin shell. It was hypothesized that pore selection and iron traffic depend on dynamic allostery with no conformational changes in the backbone. In this study, we detect the allosteric networks in Pseudomonas aeruginosa bacterioferritin (BfrB), bacterial ferritin (FtnA), and bullfrog M and L ferritins (Ftns) by a network-weaving algorithm (NWA) that passes threads of an allosteric network through highly correlated residues using hierarchical clustering. The residue-residue correlations are calculated in the packing-on elastic network model that introducesmore » atom packing into the common packing-off model. Applying NWA revealed that each of the molecules has an extended allosteric network mostly buried inside the ferritin shell. The structure of the networks is consistent with experimental observations of iron transport: The allosteric networks in BfrB and FtnA connect the ferroxidase center with the 4-fold pores and B-pores, leaving the 3-fold pores unengaged. In contrast, the allosteric network directly links the 3-fold pores with the 4-fold pores in M and L Ftns. The majority of the network residues are either on the inner surface or buried inside the subunit fold or at the subunit interfaces. We hypothesize that the ferritin structures evolved in a way to limit the influence of functionally unrelated events in the cytoplasm on the allosteric network to maintain stability of the translocation mechanisms. We showed that the residue-residue correlations and the resultant long-range cooperativity depend on the ferritin shell packing, which, in turn, depends on protein sequence composition. Switching from the packing-on to the packing-off model reduces correlations by 35%–38% so that no allosteric network can be found. The influence of the side-chain packing on the allosteric networks explains the diversity in mechanisms of iron traffic suggested by experimental approaches.« less

Structural basis for the Nanos-mediated recruitment of the CCR4–NOT complex and translational repression

PubMed Central

Bhandari, Dipankar; Raisch, Tobias; Weichenrieder, Oliver; Jonas, Stefanie; Izaurralde, Elisa

2014-01-01

The RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4–NOT deadenylase complex. Here, we show that the three human Nanos paralogs (Nanos1–3) interact with the CNOT1 C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1–3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1–3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. Our findings provide the structural basis for the recruitment of the CCR4–NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4–NOT complex as the main effector complex for Nanos function. PMID:24736845

Redox-regulated dynamic interplay between Cox19 and the copper-binding protein Cox11 in the intermembrane space of mitochondria facilitates biogenesis of cytochrome c oxidase

PubMed Central

Bode, Manuela; Woellhaf, Michael W.; Bohnert, Maria; van der Laan, Martin; Sommer, Frederik; Jung, Martin; Zimmermann, Richard; Schroda, Michael; Herrmann, Johannes M.

2015-01-01

Members of the twin Cx9C protein family constitute the largest group of proteins in the intermembrane space (IMS) of mitochondria. Despite their conserved nature and their essential role in the biogenesis of the respiratory chain, the molecular function of twin Cx9C proteins is largely unknown. We performed a SILAC-based quantitative proteomic analysis to identify interaction partners of the conserved twin Cx9C protein Cox19. We found that Cox19 interacts in a dynamic manner with Cox11, a copper transfer protein that facilitates metalation of the Cu(B) center of subunit 1 of cytochrome c oxidase. The interaction with Cox11 is critical for the stable accumulation of Cox19 in mitochondria. Cox19 consists of a helical hairpin structure that forms a hydrophobic surface characterized by two highly conserved tyrosine-leucine dipeptides. These residues are essential for Cox19 function and its specific binding to a cysteine-containing sequence in Cox11. Our observations suggest that an oxidative modification of this cysteine residue of Cox11 stimulates Cox19 binding, pointing to a redox-regulated interplay of Cox19 and Cox11 that is critical for copper transfer in the IMS and thus for biogenesis of cytochrome c oxidase. PMID:25926683

The E2 Domains of APP and APLP1 Share a Conserved Mode of Dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Lee; Y Xue; J Hulbert

2011-12-31

Amyloid precursor protein (APP) is genetically linked to Alzheimer's disease. APP is a type I membrane protein, and its oligomeric structure is potentially important because this property may play a role in its function or affect the processing of the precursor by the secretases to generate amyloid {beta}-peptide. Several independent studies have shown that APP can form dimers in the cell, but how it dimerizes remains controversial. At least three regions of the precursor, including a centrally located and conserved domain called E2, have been proposed to contribute to dimerization. Here we report two new crystal structures of E2, onemore » from APP and the other from APLP1, a mammalian APP homologue. Comparison with an earlier APP structure, which was determined in a different space group, shows that the E2 domains share a conserved and antiparallel mode of dimerization. Biophysical measurements in solution show that heparin binding induces E2 dimerization. The 2.1 {angstrom} resolution electron density map also reveals phosphate ions that are bound to the protein surface. Mutational analysis shows that protein residues interacting with the phosphate ions are also involved in heparin binding. The locations of two of these residues, Arg-369 and His-433, at the dimeric interface suggest a mechanism for heparin-induced protein dimerization.« less

Alkyl hydroperoxide reductase from Bacillus aquimaris MKSC 6.2 protects Esherichia coli from oxidative stress.

PubMed

Natalia, Dessy; Jumadila, Ozi; Anggraini, Irika Devi; Meutia, Febrina; Puspasari, Fernita; Hasan, Khomaini

2016-07-01

Alkyl hydroperoxide reductase genes (ahpCF) from the soft coral associated Bacillus aquimaris MKSC6.2 have been isolated. The cloned 546 bp ahpC gene encodes a 181 amino acid residues polypeptide. The AhpC belongs to typical 2-Cys peroxiredoxin (Prx) containing conserved peroxidatic cysteine residue (C46 ) required for hydroperoxide reduction and conserved resolving cysteine (C166 ). The isolated 1530 bp ahpF gene encodes a polypeptide of 509 amino acid residues with two conserved C128 HNC131 and C337 PHC340 catalytic residues required for reduction of oxidized-AhpC during catalytic turnover. A survival study with Escherichia coli showed that overexpression of AhpC and AhpF resulted in a total protection against 0.16 mM t-butyl hydroperoxide. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Localization of functional receptor epitopes on the structure of ciliary neurotrophic factor indicates a conserved, function-related epitope topography among helical cytokines.

PubMed

Panayotatos, N; Radziejewska, E; Acheson, A; Somogyi, R; Thadani, A; Hendrickson, W A; McDonald, N Q

1995-06-09

By rational mutagenesis, receptor-specific functional analysis, and visualization of complex formation in solution, we identified individual amino acid side chains involved specifically in the interaction of ciliary neurotrophic factor (CNTF) with CNTFR alpha and not with the beta-components, gp130 and LIFR. In the crystal structure, the side chains of these residues, which are located in helix A, the AB loop, helix B, and helix D, are surface accessible and are clustered in space, thus constituting an epitope for CNTFR alpha. By the same analysis, a partial epitope for gp130 was also identified on the surface of helix A that faces away from the alpha-epitope. Superposition of the CNTF and growth hormone structures showed that the location of these epitopes on CNTF is analogous to the location of the first and second receptor epitopes on the surface of growth hormone. Further comparison with proposed binding sites for alpha- and beta-receptors on interleukin-6 and leukemia inhibitory factor indicated that this epitope topology is conserved among helical cytokines. In each case, epitope I is utilized by the specificity-conferring component, whereas epitopes II and III are used by accessory components. Thus, in addition to a common fold, helical cytokines share a conserved order of receptor epitopes that is function related.

Identification of amino acid residues in Streptococcus mutans glucosyltransferases influencing the structure of the glucan product.

PubMed Central

Shimamura, A; Nakano, Y J; Mukasa, H; Kuramitsu, H K

1994-01-01

The glucosyltransferases (GTFs) of mutans streptococci are important virulence factors in the sucrose-dependent colonization of tooth surfaces by these organisms. To investigate the structure-function relationship of the GTFs, an approach was initiated to identify amino acid residues of the GTFs which affect the incorporation of glucose residues into the glucan polymer. Conserved amino acid residues were identified in the GTF-S and GTF-I enzymes of the mutans streptococci and were selected for site-directed mutagenesis in the corresponding enzymes from Streptococcus mutans GS5. Conversion of six amino acid residues of the GTF-I enzyme to those present at the corresponding positions in GTF-S, either singly or in multiple combinations, resulted in enzymes synthesizing increased levels of soluble glucans. The enzyme containing six alterations synthesized 73% water-soluble glucan in the absence of acceptor dextran T10, while parental enzyme GTF-I synthesized no such glucan product. Conversely, when residue 589 of the GTF-S enzyme was converted from Thr to either Asp or Glu, the resulting enzyme synthesized primarily water-insoluble glucan in the absence of the acceptor. Therefore, this approach has identified several amino acid positions which influence the nature of the glucan product synthesized by GTFs. PMID:8050997

Possible roles of exceptionally conserved residues around the selectivity filters of sodium and calcium channels.

PubMed

Tikhonov, Denis B; Zhorov, Boris S

2011-01-28

In the absence of x-ray structures of sodium and calcium channels their homology models are used to rationalize experimental data and design new experiments. A challenge is to model the outer-pore region that folds differently from potassium channels. Here we report a new model of the outer-pore region of the NaV1.4 channel, which suggests roles of highly conserved residues around the selectivity filter. The model takes from our previous study (Tikhonov, D. B., and Zhorov, B. S. (2005) Biophys. J. 88, 184-197) the general disposition of the P-helices, selectivity filter residues, and the outer carboxylates, but proposes new intra- and inter-domain contacts that support structural stability of the outer pore. Glycine residues downstream from the selectivity filter are proposed to participate in knob-into-hole contacts with the P-helices and S6s. These contacts explain the adapted tetrodotoxin resistance of snakes that feed on toxic prey through valine substitution of isoleucine in the P-helix of repeat IV. Polar residues five positions upstream from the selectivity filter residues form H-bonds with the ascending-limb backbones. Exceptionally conserved tryptophans are engaged in inter-repeat H-bonds to form a ring whose π-electrons would facilitate passage of ions from the outer carboxylates to the selectivity filter. The outer-pore model of CaV1.2 derived from the NaV1.4 model is also stabilized by the ring of exceptionally conservative tryptophans and H-bonds between the P-helices and ascending limbs. In this model, the exceptionally conserved aspartate downstream from the selectivity-filter glutamate in repeat II facilitates passage of calcium ions to the selectivity-filter ring through the tryptophan ring. Available experimental data are discussed in view of the models.

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

PubMed

Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J; Davis, Rebecca M; May, Janine M; Trauger, Sunia A; Kahne, Daniel; Ruiz, Natividad

2016-10-18

The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB 2 FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB 2 FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB 2 FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the cytoplasmic membrane prior to transport to the cell surface. How ATP hydrolysis is coupled with LPS release from the membrane is not understood. We have identified residues at the interface between the ATPase and the transmembrane domains of this heteromeric ABC complex that are important for LPS transport, some of which coordinate ATPase activity with LPS release. Copyright © 2016 Simpson et al.

A computational tool to predict the evolutionarily conserved protein-protein interaction hot-spot residues from the structure of the unbound protein.

PubMed

Agrawal, Neeraj J; Helk, Bernhard; Trout, Bernhardt L

2014-01-21

Identifying hot-spot residues - residues that are critical to protein-protein binding - can help to elucidate a protein's function and assist in designing therapeutic molecules to target those residues. We present a novel computational tool, termed spatial-interaction-map (SIM), to predict the hot-spot residues of an evolutionarily conserved protein-protein interaction from the structure of an unbound protein alone. SIM can predict the protein hot-spot residues with an accuracy of 36-57%. Thus, the SIM tool can be used to predict the yet unknown hot-spot residues for many proteins for which the structure of the protein-protein complexes are not available, thereby providing a clue to their functions and an opportunity to design therapeutic molecules to target these proteins. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Hotspots for allosteric regulation on protein surfaces

PubMed Central

Reynolds, Kimberly A.; McLaughlin, Richard N.; Ranganathan, Rama

2012-01-01

Recent work indicates a general architecture for proteins in which sparse networks of physically contiguous and co-evolving amino acids underlie basic aspects of structure and function. These networks, termed sectors, are spatially organized such that active sites are linked to many surface sites distributed throughout the structure. Using the metabolic enzyme dihydrofolate reductase as a model system, we show that (1) the sector is strongly correlated to a network of residues undergoing millisecond conformational fluctuations associated with enzyme catalysis and (2) sector-connected surface sites are statistically preferred locations for the emergence of allosteric control in vivo. Thus, sectors represent an evolutionarily conserved “wiring” mechanism that can enable perturbations at specific surface positions to rapidly initiate conformational control over protein function. These findings suggest that sectors enable the evolution of intermolecular communication and regulation. PMID:22196731

Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.

PubMed Central

Mizrahi, V; Usdin, M T; Harington, A; Dudding, L R

1990-01-01

Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity. Images PMID:1699202

Crystal Structures of Mite Allergens Der f 1 and Der p 1 Reveal Differences in Surface-Exposed Residues that May Influence Antibody Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chruszcz, Maksymilian; Chapman, Martin D.; Vailes, Lisa D.

2009-12-01

The Group 1 mite allergens, Der f 1 and Der p 1, are potent allergens excreted by Dermatophagoides farinae and Dermatophagoides pteronyssinus, respectively. The human IgE antibody responses to the Group 1 allergens show more cross-reactivity than the murine IgG antibody responses which are largely species-specific. Here, we report the crystal structure of the mature form of Der f 1, which was isolated from its natural source, and a new, high-resolution structure of mature recombinant Der p 1. Unlike Der p 1, Der f 1 is monomeric both in the crystalline state and in solution. Moreover, no metal binding ismore » observed in the structure of Der f 1, despite the fact that all amino acids involved in Ca{sup 2+} binding in Der p 1 are completely conserved in Der f 1. Although Der p 1 and Der f 1 share extensive sequence identity, comparison of the crystal structures of both allergens revealed structural features which could explain the differences in murine and human IgE antibody responses to these allergens. There are structural differences between Der f 1 and Der p 1 which are unevenly distributed on the allergens' surfaces. This uneven spatial arrangement of conserved versus altered residues could explain both the specificity and cross-reactivity of antibodies against Der f 1 and Der p 1.« less

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

USDA-ARS?s Scientific Manuscript database

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...

Dynamic Fatigue of ULE Glass

NASA Technical Reports Server (NTRS)

Tucker, Dennis S.; Nettles, Alan T.; Brantley, Lott W. (Technical Monitor)

2001-01-01

Ultra Low Expansion (ULE) glass is used in a number of applications which require a low thermal expansion coefficient. One such application is telescope mirror elements. An allowable stress can be calculated for this material based upon modulus of rupture data; however, this does not take into account the problem of delayed failure. Delayed failure, due to stress corrosion can significantly shorten the lifetime of a glass article. Knowledge of the factors governing the rate of subcritical flaw growth in a given environment enables the development of relations between lifetime, applied stress and failure probability for the material under study. Dynamic fatigue is one method of obtaining the necessary information to develop these relationships. In this study, the dynamic fatigue method was used to construct time-to-failure diagrams for both 230/270 ground and optically polished samples. The grinding and polishing process reduces the surface flaw size and subsurface damage, and relieves residual stress by removing materials with successively smaller grinding media. This resulted in an increase in the strength of the optic during the grinding and polishing sequence. There was also an increase in the lifetime due to grinding and polishing. It was found that using the fatigue parameters determined from the 230/270 grit surface are not significantly different from the optically polished values. Although the lower bound of the polished samples is more conservative, neither time-to-failure curves lie beyond the upper or lower bound of the confidence limits. Therefore, designers preferring conservative limits could use samples without residual stress present (polished samples) to determine the fatigue parameters and inert Weibull parameters from samples with the service condition surface, to determine time-to-failure of the optical element.

A novel thromboxane A2 receptor N42S variant results in reduced surface expression and platelet dysfunction.

PubMed

Nisar, Shaista P; Lordkipanidzé, Marie; Jones, Matthew L; Dawood, Ban; Murden, Sherina; Cunningham, Margaret R; Mumford, Andrew D; Wilde, Jonathan T; Watson, Steve P; Mundell, Stuart J; Lowe, Gillian C

2014-05-05

A small number of thromboxane receptor variants have been described in patients with a bleeding history that result in platelet dysfunction. We have identified a patient with a history of significant bleeding, who expresses a novel heterozygous thromboxane receptor variant that predicts an asparagine to serine substitution (N42S). This asparagine is conserved across all class A GPCRs, suggesting a vital role for receptor structure and function.We investigated the functional consequences of the TP receptor heterozygous N42S substitution by performing platelet function studies on platelet-rich plasma taken from the patient and healthy controls. We investigated the N42S mutation by expressing the wild-type (WT) and mutant receptor in human embryonic kidney (HEK) cells. Aggregation studies showed an ablation of arachidonic acid responses in the patient, whilst there was right-ward shift of the U46619 concentration response curve (CRC). Thromboxane generation was unaffected. Calcium mobilisation studies in cells lines showed a rightward shift of the U46619 CRC in N42S-expressing cells compared to WT. Radioligand binding studies revealed a reduction in BMax in platelets taken from the patient and in N42S-expressing cells, whilst cell studies confirmed poor surface expression. We have identified a novel thromboxane receptor variant, N42S, which results in platelet dysfunction due to reduced surface expression. It is associated with a significant bleeding history in the patient in whom it was identified. This is the first description of a naturally occurring variant that results in the substitution of this highly conserved residue and confirms the importance of this residue for correct GPCR function.

Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopy

PubMed Central

Li, Wei; Hoffman, David W.

2001-01-01

Translation initiation factor 1A (aIF-1A) from the archaeon Methanococcus jannaschii was expressed in Escherichia coli, purified, and characterized in terms of its structure and dynamics using multidimensional NMR methods. The protein was found to be a member of the OB-fold family of RNA-associated proteins, containing a barrel of five beta-strands, a feature that is shared with the homologous eukaryotic translation initiation factor 1A (eIF-1A), as well as the prokaryotic translation initiation factor IF1. External to the β barrel, aIF-1A contains an α-helix at its C-terminal and a flexible loop at its N-terminal, features that are qualitatively similar to those found in eIF-1A, but not present in prokaryotic IF1. The structural model of aIF-1A, when used in combination with primary sequence information for aIF-1A in divergent species, permitted the most-conserved residues on the protein surface to be identified, including the most likely candidates for direct interaction with the 16S ribosomal RNA and other components of the translational apparatus. Several of the conserved surface residues appear to be unique to the archaea. Nitrogen-15 relaxation and amide exchange rate data were used to characterize the internal motions within aIF-1A, providing evidence that the protein surfaces that are most likely to participate in intermolecular interactions are relatively flexible. A model is proposed, suggesting some specific interactions that may occur between aIF-1A and the small subunit of the archaeal ribosome. PMID:11714910

Protein structure based prediction of catalytic residues

PubMed Central

2013-01-01

Background Worldwide structural genomics projects continue to release new protein structures at an unprecedented pace, so far nearly 6000, but only about 60% of these proteins have any sort of functional annotation. Results We explored a range of features that can be used for the prediction of functional residues given a known three-dimensional structure. These features include various centrality measures of nodes in graphs of interacting residues: closeness, betweenness and page-rank centrality. We also analyzed the distance of functional amino acids to the general center of mass (GCM) of the structure, relative solvent accessibility (RSA), and the use of relative entropy as a measure of sequence conservation. From the selected features, neural networks were trained to identify catalytic residues. We found that using distance to the GCM together with amino acid type provide a good discriminant function, when combined independently with sequence conservation. Using an independent test set of 29 annotated protein structures, the method returned 411 of the initial 9262 residues as the most likely to be involved in function. The output 411 residues contain 70 of the annotated 111 catalytic residues. This represents an approximately 14-fold enrichment of catalytic residues on the entire input set (corresponding to a sensitivity of 63% and a precision of 17%), a performance competitive with that of other state-of-the-art methods. Conclusions We found that several of the graph based measures utilize the same underlying feature of protein structures, which can be simply and more effectively captured with the distance to GCM definition. This also has the added the advantage of simplicity and easy implementation. Meanwhile sequence conservation remains by far the most influential feature in identifying functional residues. We also found that due the rapid changes in size and composition of sequence databases, conservation calculations must be recalibrated for specific reference databases. PMID:23433045

1H, 13C and 15N assignment of the C-terminal domain of GNA2132 from Neisseria meningitidis.

PubMed

Esposito, Veronica; Musi, Valeria; Veggi, Daniele; Pastore, Annalisa; Pizza, Mariagrazia

2010-04-01

GNA2132 (Genome-derived Neisseria Antigen 2132) is a surface-exposed lipoprotein discovered by reverse vaccinology and expressed by genetically diverse Neisseria meningitidis strains (Pizza et al. 2000). The protein induces bactericidal antibodies against most strains of Meningococccus and has been included in a multivalent recombinant vaccine against N. meningitidis serogroup B. Structure determination of GNA2132 is important for understanding the antigenic properties of the protein in view of increased efficiency vaccine development. We report practically complete (1)H, (13)C and (15)N assignment of the detectable spectrum of a highly conserved C-terminal region of GNA2132 (residues 245-427) in micellar solution, a medium used to improve the spectral quality. The first 32 residues of our construct up to residue 277 were not visible in the spectrum, presumably because of line broadening due to solvent and/or conformational exchange. Secondary structure predictions based on chemical shift information indicate the presence of an all beta-protein with eight beta strands.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

PubMed Central

Du, Yushen; Wu, Nicholas C.; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting

2016-01-01

ABSTRACT Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. PMID:27803181

Vegetation effects on soil water erosion rates and nutrient losses at Santa Catarina highlands, south Brazil

NASA Astrophysics Data System (ADS)

Bertol, I.; Barbosa, F. T.; Vidal Vázquez, E.; Paz Ferreiro, J.

2009-04-01

Water erosion involves three main processes: detachment, transport and deposition of soil particles. The main factors affecting water erosion are rainfall, soil, topography, soil management and land cover and use. Soil erosion potential is increased if the soil has no or very little vegetative cover of plants and/or crop residues, whereas plant and residue cover substantially decrease rates of soil erosion. Plant and residue cover protects the soil from raindrop impact and splash, tends to slow down the movement of surface runoff and allows excess surface water to infiltrate. Moreover, plant and residue cover improve soil physical, chemical and biological properties. Soils with improved structure have a greater resistance to erosion. By contrast, accelerated soil erosion is accentuated by deforestation, biomass burning, plowing and disking, cultivation of open-row crops, etc. The erosion-reducing effectiveness of plant and/or residue covers depends on the type, extent and quantity of cover. Vegetation and residue combinations that completely cover the soil are the most efficient in controlling soil. Partially incorporated residues and residual roots are also important, as these provide channels that allow surface water to move into the soil. The effectiveness of any crop, management system or protective cover also depends on how much protection is available at various periods during the year, relative to the amount of erosive rainfall that falls during these periods. Most of the erosion on annual row crop land can be reduced by leaving a residue cover greater after harvest and over the winter months, or by inter-seeding a forage crop. Soil erosion potential is also affected by tillage operations and tillage system. Conservation tillage reduces water erosion in relation to conventional tillage by increasing soil cover and soil surface roughness. Here, we review the effect of vegetation on soil erosion in the Santa Catarina highlands, south of Brazil, under subtropical climatic conditions. The area cropped under no tillage in Brazil has increased rapidly since 1990, especially in the southern region. This practice was first introduced in the 1970s as a strategy to control soil erosion and continuous declines in land productivity under conventional tillage systems. No tillage almost entirely keeps the previous crop residue on the surface. In the last 15 years soil and water losses by water erosion have been quantified for different soil tillage systems, diverse crop rotations and successive crop stages under simulated and natural rain conditions. Plot experiments showed that soil losses under no tillage systems with a vegetative cover were 98% lower when compared with conventionally tilled bare soil. Moreover water losses were 60% lower for these conditions. Conventional tillage (plowing + harrowing) in the presence of vegetative cover reduced soil losses and water losses by 80% and 50%, respectively, taken the uncultivated bare soil as a reference. The review includes the effect of vegetative cover on nutrient losses at the studied sites in the Santa Catarina highlands.

Grass mulching effect on infiltration, surface runoff and soil loss of three agricultural soils in Nigeria.

PubMed

Adekalu, K O; Olorunfemi, I A; Osunbitan, J A

2007-03-01

Mulching the soil surface with a layer of plant residue is an effective method of conserving water and soil because it reduces surface runoff, increases infiltration of water into the soil and retard soil erosion. The effectiveness of using elephant grass (Pennisetum purpureum) as mulching material was evaluated in the laboratory using a rainfall simulator set at rainfall intensities typical of the tropics. Six soil samples, two from each of the three major soil series representing the main agricultural soils in South Western Nigeria were collected, placed on three different slopes, and mulched with different rates of the grass. The surface runoff, soil loss, and apparent cumulative infiltration were then measured under each condition. The results with elephant grass compared favorably with results from previous experiments using rice straw. Runoff and soil loss decreased with the amount of mulch used and increased with slope. Surface runoff, infiltration and soil loss had high correlations (R = 0.90, 0.89, and 0.86, respectively) with slope and mulch cover using surface response analysis. The mean surface runoff was correlated negatively with sand content, while mean soil loss was correlated positively with colloidal content (clay and organic matter) of the soil. Infiltration was increased and soil loss was reduced greatly with the highest cover. Mulching the soils with elephant grass residue may benefit late cropping (second cropping) by increasing stored soil water for use during dry weather and help to reduce erosion on sloping land.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R., E-mail: grw7@cornell.ed

The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined withmore » cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.« less

Hydration in drug design. 3. Conserved water molecules at the ligand-binding sites of homologous proteins

NASA Astrophysics Data System (ADS)

Poornima, C. S.; Dean, P. M.

1995-12-01

Water molecules are known to play an important rôle in mediating protein-ligand interactions. If water molecules are conserved at the ligand-binding sites of homologous proteins, such a finding may suggest the structural importance of water molecules in ligand binding. Structurally conserved water molecules change the conventional definition of `binding sites' by changing the shape and complementarity of these sites. Such conserved water molecules can be important for site-directed ligand/drug design. Therefore, five different sets of homologous protein/protein-ligand complexes have been examined to identify the conserved water molecules at the ligand-binding sites. Our analysis reveals that there are as many as 16 conserved water molecules at the FAD binding site of glutathione reductase between the crystal structures obtained from human and E. coli. In the remaining four sets of high-resolution crystal structures, 2-4 water molecules have been found to be conserved at the ligand-binding sites. The majority of these conserved water molecules are either bound in deep grooves at the protein-ligand interface or completely buried in cavities between the protein and the ligand. All these water molecules, conserved between the protein/protein-ligand complexes from different species, have identical or similar apolar and polar interactions in a given set. The site residues interacting with the conserved water molecules at the ligand-binding sites have been found to be highly conserved among proteins from different species; they are more conserved compared to the other site residues interacting with the ligand. These water molecules, in general, make multiple polar contacts with protein-site residues.

Measurement of the fluorescence of crop residues: A tool for controlling soil erosion

NASA Technical Reports Server (NTRS)

Daughtry, C. S. T.; Mcmurtrey, J. E., III; Chappelle, E. W.; Hunter, W. J.

1994-01-01

Management of crop residues, the portion of a crop left in the field after harvest, is an important conservation practice for minimizing soil erosion and for improving water quality. Quantification of crop residue cover is required to evaluate the effectiveness of conservation tillage practices. Methods are needed to quantify residue cover that are rapid, accurate, and objective. The fluorescence of crop residue was found to be a broadband phenomenon with emission maxima at 420 to 495 nm for excitations of 350 to 420 nm. Soils had low intensity broadband emissions over the 400 to 690 nm region for excitations of 300 to 600 nm. The range of relative fluorescence intensities for the crop residues was much greater than the fluorescence observed of the soils. As the crop residues decompose their blue fluorescence values approach the fluorescence of the soil. Fluorescence techniques are concluded to be less ambiguous and better suited for discriminating crop residues and soils than reflectance methods. If properly implemented, fluorescence techniques can be used to quantify, not only crop residue cover, but also photosynthetic efficiency in the field.

Mutations in the Cytoplasmic Domain of the Newcastle Disease Virus Fusion Protein Confer Hyperfusogenic Phenotypes Modulating Viral Replication and Pathogenicity

PubMed Central

Samal, Sweety; Khattar, Sunil K.; Paldurai, Anandan; Palaniyandi, Senthilkumar; Zhu, Xiaoping; Collins, Peter L.

2013-01-01

The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis. PMID:23843643

Mutations in the cytoplasmic domain of the Newcastle disease virus fusion protein confer hyperfusogenic phenotypes modulating viral replication and pathogenicity.

PubMed

Samal, Sweety; Khattar, Sunil K; Paldurai, Anandan; Palaniyandi, Senthilkumar; Zhu, Xiaoping; Collins, Peter L; Samal, Siba K

2013-09-01

The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis.

Construction and first characterization of two reciprocal hybrids between LamB from Escherichia coli K12 and Klebsiella pneumoniae.

PubMed

Werts, C; Charbit, A

1993-05-01

The LamB proteins from Klebsiella pneumoniae and Escherichia coli K12 were previously shown to be highly homologous. The most conserved parts correspond to the N-proximal third and to the transmembranous portions of the molecule, while the variability occurred essentially within regions exposed to the cell surface or to the periplasm. Since the two proteins displayed identical in vitro trimer stability and in vivo pore properties, we tested whether the N-terminal parts of the two proteins could be exchanged and still allow the formation of stable and functional maltoporins. For that purpose, we expressed the LamB protein from K. pneumoniae in E. coli K12, and constructed two reciprocal hybrids between LamB from E. coli K12 and LamB from K. pneumoniae. The first hybrid (LamBE.c.-K.p.) is composed of residues 1 to 183 from LamBE.c. followed by residues 184 to 404 from LamBK.p. The second one comprises residues 1 to 183 from LamBK.p., followed by residues 184 to 421 from LamBE.c. (LamBK.p.-E.c.). Both hybrid proteins were correctly incorporated in the outer membrane of E. coli K12. Like the two parental LamB proteins, the two hybrids could be purified by affinity chromatography on a starch-sepharose column. The LamBE.c.-K.p. hybrid formed highly stable trimers, but was strongly impaired in its in vivo maltose transport function (15% of the wild-type level). The trimers formed by LamBK.p.-E.c. hybrid were less stable, but could be detected on the surface of intact cells by four anti-LamBE.c. monoclonal antibodies. This hybrid was also affected in its in vivo maltose transport function (30% of the wild-type level). As expected from the location of the residues critical for phage adsorption, both proteins had lost the phage receptor activity of the E. coli K12 LamB protein. We also examined whether LamBE.c. could form heterotrimers with LamBK.p., LamBK.p.-E.c., and LamBE.c.-K.p. In no case were heterotrimers detected, indicating that both terminal parts of the LamB protein are involved in homotrimer formation. All these data suggest that trimer formation and activity involve rare variable residues in the conserved regions and/or variable regions.

A RecA Protein Surface Required for Activation of DNA Polymerase V

PubMed Central

Gruber, Angela J.; Erdem, Aysen L.; Sabat, Grzegorz; Karata, Kiyonobu; Jaszczur, Malgorzata M.; Vo, Dan D.; Olsen, Tayla M.; Woodgate, Roger; Goodman, Myron F.; Cox, Michael M.

2015-01-01

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis. PMID:25811184

Structure of deformed wing virus, a major honey bee pathogen.

PubMed

Škubník, Karel; Nováček, Jiří; Füzik, Tibor; Přidal, Antonín; Paxton, Robert J; Plevka, Pavel

2017-03-21

The worldwide population of western honey bees ( Apis mellifera ) is under pressure from habitat loss, environmental stress, and pathogens, particularly viruses that cause lethal epidemics. Deformed wing virus (DWV) from the family Iflaviridae , together with its vector, the mite Varroa destructor , is likely the major threat to the world's honey bees. However, lack of knowledge of the atomic structures of iflaviruses has hindered the development of effective treatments against them. Here, we present the virion structures of DWV determined to a resolution of 3.1 Å using cryo-electron microscopy and 3.8 Å by X-ray crystallography. The C-terminal extension of capsid protein VP3 folds into a globular protruding (P) domain, exposed on the virion surface. The P domain contains an Asp-His-Ser catalytic triad that is, together with five residues that are spatially close, conserved among iflaviruses. These residues may participate in receptor binding or provide the protease, lipase, or esterase activity required for entry of the virus into a host cell. Furthermore, nucleotides of the DWV RNA genome interact with VP3 subunits. The capsid protein residues involved in the RNA binding are conserved among honey bee iflaviruses, suggesting a putative role of the genome in stabilizing the virion or facilitating capsid assembly. Identifying the RNA-binding and putative catalytic sites within the DWV virion structure enables future analyses of how DWV and other iflaviruses infect insect cells and also opens up possibilities for the development of antiviral treatments.

The perturbation of tryptophan fluorescence by phenylalanine to alanine mutations identifies the hydrophobic core in a subset of bacterial Ig-like domains.

PubMed

Raman, Rajeev; Ptak, Christopher P; Hsieh, Ching-Lin; Oswald, Robert E; Chang, Yung-Fu; Sharma, Yogendra

2013-07-09

Many host-parasite interactions are mediated via surface-exposed proteins containing bacterial immunoglobulin-like (Big) domains. Here, we utilize the spectral properties of a conserved Trp to provide evidence that, along with a Phe, these residues are positioned within the hydrophobic core of a subset of Big_2 domains. The mutation of the Phe to Ala decreases Big_2 domain stability and impairs the ability of LigBCen2 to bind to the host protein, fibronectin.

Identification and Analysis of Novel Amino-Acid Sequence Repeats in Bacillus anthracis str. Ames Proteome Using Computational Tools

PubMed Central

Hemalatha, G. R.; Rao, D. Satyanarayana; Guruprasad, L.

2007-01-01

We have identified four repeats and ten domains that are novel in proteins encoded by the Bacillus anthracis str. Ames proteome using automated in silico methods. A “repeat” corresponds to a region comprising less than 55-amino-acid residues that occur more than once in the protein sequence and sometimes present in tandem. A “domain” corresponds to a conserved region with greater than 55-amino-acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 57-amino-acid-residue PxV domain, (2) 122-amino-acid-residue FxF domain, (3) 111-amino-acid-residue YEFF domain, (4) 109-amino-acid-residue IMxxH domain, (5) 103-amino-acid-residue VxxT domain, (6) 84-amino-acid-residue ExW domain, (7) 104-amino-acid-residue NTGFIG domain, (8) 36-amino-acid-residue NxGK repeat, (9) 95-amino-acid-residue VYV domain, (10) 75-amino-acid-residue KEWE domain, (11) 59-amino-acid-residue AFL domain, (12) 53-amino-acid-residue RIDVK repeat, (13) (a) 41-amino-acid-residue AGQF repeat and (b) 42-amino-acid-residue GSAL repeat. A repeat or domain type is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure. PMID:17538688

A conserved mechanism for replication origin recognition and binding in archaea.

PubMed

Majerník, Alan I; Chong, James P J

2008-01-15

To date, methanogens are the only group within the archaea where firing DNA replication origins have not been demonstrated in vivo. In the present study we show that a previously identified cluster of ORB (origin recognition box) sequences do indeed function as an origin of replication in vivo in the archaeon Methanothermobacter thermautotrophicus. Although the consensus sequence of ORBs in M. thermautotrophicus is somewhat conserved when compared with ORB sequences in other archaea, the Cdc6-1 protein from M. thermautotrophicus (termed MthCdc6-1) displays sequence-specific binding that is selective for the MthORB sequence and does not recognize ORBs from other archaeal species. Stabilization of in vitro MthORB DNA binding by MthCdc6-1 requires additional conserved sequences 3' to those originally described for M. thermautotrophicus. By testing synthetic sequences bearing mutations in the MthORB consensus sequence, we show that Cdc6/ORB binding is critically dependent on the presence of an invariant guanine found in all archaeal ORB sequences. Mutation of a universally conserved arginine residue in the recognition helix of the winged helix domain of archaeal Cdc6-1 shows that specific origin sequence recognition is dependent on the interaction of this arginine residue with the invariant guanine. Recognition of a mutated origin sequence can be achieved by mutation of the conserved arginine residue to a lysine or glutamine residue. Thus despite a number of differences in protein and DNA sequences between species, the mechanism of origin recognition and binding appears to be conserved throughout the archaea.

[Conserved motifs in voltage sensing proteins].

PubMed

Wang, Chang-He; Xie, Zhen-Li; Lv, Jian-Wei; Yu, Zhi-Dan; Shao, Shu-Li

2012-08-25

This paper was aimed to study conserved motifs of voltage sensing proteins (VSPs) and establish a voltage sensing model. All VSPs were collected from the Uniprot database using a comprehensive keyword search followed by manual curation, and the results indicated that there are only two types of known VSPs, voltage gated ion channels and voltage dependent phosphatases. All the VSPs have a common domain of four helical transmembrane segments (TMS, S1-S4), which constitute the voltage sensing module of the VSPs. The S1 segment was shown to be responsible for membrane targeting and insertion of these proteins, while S2-S4 segments, which can sense membrane potential, for protein properties. Conserved motifs/residues and their functional significance of each TMS were identified using profile-to-profile sequence alignments. Conserved motifs in these four segments are strikingly similar for all VSPs, especially, the conserved motif [RK]-X(2)-R-X(2)-R-X(2)-[RK] was presented in all the S4 segments, with positively charged arginine (R) alternating with two hydrophobic or uncharged residues. Movement of these arginines across the membrane electric field is the core mechanism by which the VSPs detect changes in membrane potential. The negatively charged aspartate (D) in the S3 segment is universally conserved in all the VSPs, suggesting that the aspartate residue may be involved in voltage sensing properties of VSPs as well as the electrostatic interactions with the positively charged residues in the S4 segment, which may enhance the thermodynamic stability of the S4 segments in plasma membrane.

The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

PubMed

Wieczorek, Anna; McHenry, Charles S

2006-05-05

The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

Conserved Regions of Gonococcal TbpB Are Critical for Surface Exposure and Transferrin Iron Utilization

PubMed Central

Ostberg, Karen L.; DeRocco, Amanda J.; Mistry, Shreni D.; Dickinson, Mary Kathryne

2013-01-01

The transferrin-binding proteins TbpA and TbpB enable Neisseria gonorrhoeae to obtain iron from human transferrin. The lipoprotein TbpB facilitates, but is not strictly required for, TbpA-mediated iron acquisition. The goal of the current study was to determine the contribution of two conserved regions within TbpB to the function of this protein. Using site-directed mutagenesis, the first mutation we constructed replaced the lipobox (LSAC) of TbpB with a signal I peptidase cleavage site (LAAA), while the second mutation deleted a conserved stretch of glycine residues immediately downstream of the lipobox. We then evaluated the resulting mutants for effects on TbpB expression, surface exposure, and transferrin iron utilization. Western blot analysis and palmitate labeling indicated that the lipobox, but not the glycine-rich motif, is required for lipidation of TbpB and tethering to the outer membrane. TbpB was released into the supernatant by the mutant that produces TbpB LSAC. Neither mutation disrupted the transport of TbpB across the bacterial cell envelope. When these mutant TbpB proteins were produced in a strain expressing a form of TbpA that requires TbpB for iron acquisition, growth on transferrin was either abrogated or dramatically diminished. We conclude that surface tethering of TbpB is required for optimal performance of the transferrin iron acquisition system, while the presence of the polyglycine stretch near the amino terminus of TbpB contributes significantly to transferrin iron transport function. Overall, these results provide important insights into the functional roles of two conserved motifs of TbpB, enhancing our understanding of this critical iron uptake system. PMID:23836816

An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

2016-06-13

ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface inmore » which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.« less

Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision

PubMed Central

Kan, Fengling; Davidson, Mari K.; Wahls, Wayne P.

2011-01-01

In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein–protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics. PMID:21030440

Dust-associated microbiomes from dryland wheat fields differ with tillage practice and biosolids application

NASA Astrophysics Data System (ADS)

Schlatter, Daniel C.; Schillinger, William F.; Bary, Andy I.; Sharratt, Brenton; Paulitz, Timothy C.

2018-07-01

Wind erosion is a significant threat to the productivity and sustainability of agricultural soils. In the dryland winter wheat (Triticum aestivum L.)-fallow region of Inland Pacific Northwest of the USA (PNW), farmers increasingly use conservation tillage practices to control wind erosion. In addition, some farmers in this dry region apply municipal biosolids to soils as fertilizer and a source of stable organic matter. The impacts of soil management practices on emissions of dust microbiota to the atmosphere are understudied. We used high-throughput DNA sequencing to examine the impacts of conservation tillage and biosolids amendments on the transport of dust-associated fungal and bacterial communities during simulated high-wind events over two years at Lind, WA. The fungal and bacterial communities contained in windblown dust differed significantly with tillage (conservation vs. conventional) and fertilizer (synthetic vs. biosolids) treatments. However, the richness and diversity of fungal and bacterial communities of dust did not vary significantly with tillage or fertilizer treatments. Taxa enriched in dust from fields under conservation tillage represented many plant-associated taxa that likely grow on residue left on the soil surface, whereas taxa that were more abundant with conventional tillage were those that likely grow on buried plant residue. Dust from biosolids-amended fields harbored greater abundances of taxa that likely feed on introduced carbon. Most human-associated taxa that may pose a health risk were not present in dust after biosolids amendment, although members of Clostridiaceae were enriched with this treatment. Results show that tillage and fertilizer management practices impact the composition of bioaerosols emitted during high-wind events and have potential implications for plant and human health.

Crystal Structure of the Heterotrimeric Integrin-Binding Region of Laminin-111.

PubMed

Pulido, David; Hussain, Sadaf-Ahmahni; Hohenester, Erhard

2017-03-07

Laminins are cell-adhesive glycoproteins that are essential for basement membrane assembly and function. Integrins are important laminin receptors, but their binding site on the heterotrimeric laminins is poorly defined structurally. We report the crystal structure at 2.13 Å resolution of a minimal integrin-binding fragment of mouse laminin-111, consisting of ∼50 residues of α1β1γ1 coiled coil and the first three laminin G-like (LG) domains of the α1 chain. The LG domains adopt a triangular arrangement, with the C terminus of the coiled coil situated between LG1 and LG2. The critical integrin-binding glutamic acid residue in the γ1 chain tail is surface exposed and predicted to bind to the metal ion-dependent adhesion site in the integrin β1 subunit. Additional contacts to the integrin are likely to be made by the LG1 and LG2 surfaces adjacent to the γ1 chain tail, which are notably conserved and free of obstructing glycans. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Effects of site-directed mutagenesis of mglA on motility and swarming of Myxococcus xanthus

PubMed Central

2010-01-01

Background The mglA gene from the bacterium Myxococcus xanthus encodes a 22kDa protein related to the Ras superfamily of monomeric GTPases. MglA is required for the normal function of A-motility (adventurous), S-motility (social), fruiting body morphogenesis, and sporulation. MglA and its homologs differ from all eukaryotic and other prokaryotic GTPases because they have a threonine (Thr78) in place of the highly conserved aspartate residue of the consensus PM3 (phosphate-magnesium binding) region. To identify residues critical for MglA function or potential protein interactions, and explore the function of Thr78, the phenotypes of 18 mglA mutants were characterized. Results Nine mutants, with mutations predicted to alter residues that bind the guanine base or coordinate magnesium, did not produce detectable MglA. As expected, these mutants were mot- dev- because MglA is essential for these processes. Of the remaining nine mutants, seven showed a wild-type distribution pattern for MglA but fell into two categories with regard to function. Five of the seven mutants exhibited mild phenotypes, but two mutants, T78D and P80A, abolished motility and development. The localization pattern of MglA was abolished in two mutants that were mot- spo- and dev-. These two mutants were predicted to alter surface residues at Asp52 and Thr54, which suggests that these residues are critical for proper localization and may define a protein interaction site. Improving the consensus match with Ras at Thr78 abolished function of MglA. Only the conservative serine substitution was tolerated at this position. Merodiploid constructs revealed that a subset of alleles, including mglAD52A, were dominant and also illustrated that changing the balance of MglA and its co-transcribed partner, MglB, affects A-motility. Conclusion Our results suggest that GTP binding is critical for stability of MglA because MglA does not accumulate in mutants that cannot bind GTP. The threonine in PM3 of MglA proteins represents a novel modification of the highly conserved GTPase consensus at this position. The requirement for a hydroxyl group at this position may indicate that MglA is subject to modification under certain conditions. Proper localization of MglA is critical for both motility and development and likely involves protein interactions mediated by residues Asp52 and Thr54. PMID:21083931

Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A.

2012-08-21

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the humanmore » protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.« less

Analysis of inter-residue contacts reveals folding stabilizers in P-loops of potassium, sodium, and TRPV channels.

PubMed

Korkosh, V S; Zhorov, B S; Tikhonov, D B

2016-05-01

The family of P-loop channels includes potassium, sodium, calcium, cyclic nucleotide-gated and TRPV channels, as well as ionotropic glutamate receptors. Despite vastly different physiological and pharmacological properties, the channels have structurally conserved folding of the pore domain. Furthermore, crystallographic data demonstrate surprisingly similar mutual disposition of transmembrane and membrane-diving helices. To understand determinants of this conservation, here we have compared available high-resolution structures of sodium, potassium, and TRPV1 channels. We found that some residues, which are in matching positions of the sequence alignment, occur in different positions in the 3D alignment. Surprisingly, we found 3D mismatches in well-packed P-helices. Analysis of energetics of individual residues in Monte Carlo minimized structures revealed cyclic patterns of energetically favorable inter- and intra-subunit contacts of P-helices with S6 helices. The inter-subunit contacts are rather conserved in all the channels, whereas the intra-subunit contacts are specific for particular types of the channels. Our results suggest that these residue-residue contacts contribute to the folding stabilization. Analysis of such contacts is important for structural and phylogenetic studies of homologous proteins.

Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications

PubMed Central

Cvicek, Vaclav; Goddard, William A.; Abrol, Ravinder

2016-01-01

The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs. These residues can be used to make testable hypotheses about the structural basis of receptor function and about the molecular basis of disease-associated single nucleotide polymorphisms. PMID:27028541

Roles of three amino acids of capsid proteins in mink enteritis parvovirus replication.

PubMed

Mao, Yaping; Su, Jun; Wang, Jigui; Zhang, Xiaomei; Hou, Qiang; Bian, Dawei; Liu, Weiquan

2016-08-15

Virulent mink enteritis parvovirus (MEV) strain MEV-LHV replicated to higher titers in feline F81 cells than attenuated strain MEV-L. Phylogenetic and sequence analyses of the VP2 gene of MEV-LHV, MEV-L and other strains in GenBank revealed two evolutionary branches separating virulent and attenuated strains. Three residues, 101, 232 and 411, differed between virulent and attenuated strains but were conserved within the two branches. Site-directed mutagenesis of the VP2 gene of infectious plasmids of attenuated strain MEV-L respectively replacing residues 101 Ile and 411 Ala with Thr and Glu of virulent strains (MEV-L I101T and MEV-L A411E) increased replication efficiency but still to lower levels than MEV-LHV. However, viruses with mutation of residue 232 (MEV-L I232V and MEV-L I101T/I232V/A411E) decreased viral transcription and replication levels. The three VP2 residues 101, 232 and 411, located on or near the capsid surface, played different roles in the infection processes of MEV. Copyright © 2016 Elsevier B.V. All rights reserved.

The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage.

PubMed Central

Grasby, J A; Jonathan, P; Butler, G; Gait, M J

1993-01-01

The synthesis is described of oligoribonucleotides containing the modified nucleoside O6-methylguanosine. Solid-phase oligoribonucleotide assembly was carried out by use of 2'-silyl-protected nucleoside phosphoramidites, a new O6-methylguanosine-containing synthon and a mild deprotection method. The O6-methylguanosine-modified oligonucleotides were used in the study of the role of conserved residues G5, G8 and G12 in hammerhead ribozyme cleavage. Hammerheads thus substituted at any of these positions showed an approximately 75-fold reduction in kcat whereas Km was unaffected. Hammerheads with modifications at G5 or G8 showed a significant reduction in magnesium binding affinity whereas modification at G12 had no effect. The results show that the three conserved G residues play crucial but different role sin hammerhead cleavage. PMID:8233777

Laboratory experiment on the 3D tide-induced Lagrangian residual current using the PIV technique

NASA Astrophysics Data System (ADS)

Chen, Yang; Jiang, Wensheng; Chen, Xu; Wang, Tao; Bian, Changwei

2017-12-01

The 3D structure of the tide-induced Lagrangian residual current was studied using the particle image velocimetry (PIV) technique in a long shallow narrow tank in the laboratory. At the mouth of the tank, a wave generator was used to make periodic wave which represents the tide movement, and at the head of the tank, a laterally sloping topography with the length of one fifth of the water tank was installed, above which the tide-induced Lagrangian residual current was studied. Under the weakly nonlinear condition in the present experiment setup, the results show that the Lagrangian residual velocity (LRV) field has a three-layer structure. The residual current flows inwards (towards the head) in the bottom layer and flows outwards in the middle layer, while in the surface layer, it flows inwards along the shallow side of the sloping topography and outwards along the deep side. The depth-averaged and breadth-averaged LRV are also analyzed based on the 3D LRV observations. Our results are in good agreement with the previous experiment studies, the analytical solutions with similar conditions and the observational results in real bays. Moreover, the volume flux comparison between the Lagrangian and Eulerian residual currents shows that the Eulerian residual velocity violates the mass conservation law while the LRV truly represents the inter-tidal water transport. This work enriches the laboratory studies of the LRV and offers valuable references for the LRV studies in real bays.

Polyester synthases: natural catalysts for plastics.

PubMed Central

Rehm, Bernd H A

2003-01-01

Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with a conserved cysteine residue as catalytic nucleophile. This review provides a survey of the known biochemical features of these unique enzymes and their proposed catalytic mechanism. PMID:12954080

Surface charge engineering of a Bacillus gibsonii subtilisin protease.

PubMed

Jakob, Felix; Martinez, Ronny; Mandawe, John; Hellmuth, Hendrik; Siegert, Petra; Maurer, Karl-Heinz; Schwaneberg, Ulrich

2013-08-01

In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general. In this work, three criteria for selecting amino acid substitutions of the deamidation type in the Bacillus gibsonii alkaline protease (BgAP) are proposed and systematically studied in their influence on pH-dependent activity and thermal resistance. Out of 113 possible surface amino acids, 18 (11 Asn and 7 Gln) residues of BgAP were selected and evaluated based on three proposed criteria: (1) The Asn or Gln residues should not be conserved, (2) should be surface exposed, and (3) neighbored by glycine. "Deamidation" in five (N97, N253, Q37, Q200, and Q256) out of eight (N97, N154, N250, N253, Q37, Q107, Q200, and Q256) amino acids meeting all criteria resulted in increased proteolytic activity. In addition, pH activity profiles of the variants N253D and Q256E and the combined variant N253DQ256E were dramatically shifted towards higher activity at lower pH (range of 8.5-10). Variant N253DQ256E showed twice the specific activity of wild-type BgAP and its thermal resistance increased by 2.4 °C at pH 8.5. These property changes suggest that mimicking surface deamidation by substituting Gln by Glu and/or Asn by Asp might be a simple and fast protein reengineering approach for modulating enzyme properties such as activity, pH optimum, and thermal resistance.

Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases.

PubMed

Miller, Bradley R; Sundlov, Jesse A; Drake, Eric J; Makin, Thomas A; Gulick, Andrew M

2014-10-01

Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation-PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. © 2014 Wiley Periodicals, Inc.

Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis.

PubMed

Mori, Hiroyuki; Sakashita, Sohei; Ito, Jun; Ishii, Eiji; Akiyama, Yoshinori

2018-02-23

VemP ( V ibrio protein e xport m onitoring p olypeptide) is a secretory protein comprising 159 amino acid residues, which functions as a secretion monitor in Vibrio and regulates expression of the downstream V.secDF2 genes. When VemP export is compromised, its translation specifically undergoes elongation arrest at the position where the Gln 156 codon of vemP encounters the P-site in the translating ribosome, resulting in up-regulation of V.SecDF2 production. Although our previous study suggests that many residues in a highly conserved C-terminal 20-residue region of VemP contribute to its elongation arrest, the exact role of each residue remains unclear. Here, we constructed a reporter system to easily and exactly monitor the in vivo arrest efficiency of VemP. Using this reporter system, we systematically performed a mutational analysis of the 20 residues (His 138 -Phe 157 ) to identify and characterize the arrest motif. Our results show that 15 residues in the conserved region participate in elongation arrest and that multiple interactions between important residues in VemP and in the interior of the exit tunnel contribute to the elongation arrest of VemP. The arrangement of these important residues induced by specific secondary structures in the ribosomal tunnel is critical for the arrest. Pro scanning analysis of the preceding segment (Met 120 -Phe 137 ) revealed a minor role of this region in the arrest. Considering these results, we conclude that the arrest motif in VemP is mainly composed of the highly conserved multiple residues in the C-terminal region. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystal structure of botulinum neurotoxin type A in complex with the cell surface co-receptor GT1b-insight into the toxin-neuron interaction.

PubMed

Stenmark, Pål; Dupuy, Jérôme; Imamura, Akihiro; Kiso, Makoto; Stevens, Raymond C

2008-08-15

Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in the toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.

A highly Conserved Aspartic Acid Residue of the Chitosanase from Bacillus Sp. TS Is Involved in the Substrate Binding.

PubMed

Zhou, Zhanping; Zhao, Shuangzhi; Liu, Yang; Chang, Zhengying; Ma, Yanhe; Li, Jian; Song, Jiangning

2016-11-01

The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.

Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation

PubMed Central

Caba, Cody; Ali Khan, Hyder; Auld, Janeen; Ushioda, Ryo; Araki, Kazutaka; Nagata, Kazuhiro; Mutus, Bulent

2018-01-01

Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys57 and Lys401 of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys57 and acLys401. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity. PMID:29541639

An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation.

PubMed

Friedt, Jenna; Leavens, Fern M V; Mercier, Evan; Wieden, Hans-Joachim; Kothe, Ute

2014-04-01

Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB's catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.

An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation

PubMed Central

Friedt, Jenna; Leavens, Fern M. V.; Mercier, Evan; Wieden, Hans-Joachim; Kothe, Ute

2014-01-01

Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism. PMID:24371284

The dark side of the ring: role of the DNA sliding surface of PCNA.

PubMed

De March, Matteo; De Biasio, Alfredo

2017-12-01

The proliferating cell nuclear antigen (PCNA) sliding clamp lies at the heart of the accurate duplication of eukaryotic genomes. While the outer surface of the PCNA ring interacts with polymerases and other factors, the role of the inner wall facing the DNA is elusive. Recent evidence shows that conserved basic residues in the PCNA central channel create a specific surface that recognizes the DNA backbone and enables the clamp to slide by rotationally tracking the DNA helix. The sliding surface can be modulated (i) through lysine acetylation, which triggers PCNA degradation during nucleotide excision repair (NER) and stimulates repair by homologous recombination (HR) or (ii) through binding of the protein factor p15 PAF , which turns off DNA lesion bypass. Thus, the inner surface of PCNA is unexpectedly highly regulated to control resistance to DNA damage. From a structural viewpoint, we reflect on these findings that open a new perspective on PCNA function and offer opportunities to develop tools to manipulate the DNA damage response in cancer treatment.

A conserved threonine in the S1-S2 loop of KV7.2 and K V7.3 channels regulates voltage-dependent activation.

PubMed

Füll, Yvonne; Seebohm, Guiscard; Lerche, Holger; Maljevic, Snezana

2013-06-01

The voltage-gated potassium channels KV7.2 and KV7.3 (KCNQ2/3 genes) play an important role in regulating neuronal excitability. More than 50 KCNQ2/3 mutations have been identified to cause an inherited form of epilepsy in newborns. For two of those (E119G and S122L) found in the S1-S2 region of KV7.2, we previously showed a decreased channel availability mainly at action potential subthreshold voltages caused by a slight depolarizing shift of the activation curve. Interestingly, recent studies revealed that a threonine residue within the S1-S2 loop, highly conserved among different classes of KV channels, is crucial for both their function and surface expression. To investigate the functional role of the homologous threonine residues in KV7.2 (T114) and KV7.3 (T144) channels, we replaced them with alanine and examined the electrophysiological properties using heterologous expression in CHO cells and whole cell patch clamping. Channels comprising mutant subunits yielded decreased potassium currents with slowed activation and accelerated deactivation kinetics. However, the most striking effect was a depolarizing shift in the voltage dependence of activation reaching +30 mV upon co-expression of both mutant subunits. Potential interactions of T114 within the channel were analyzed by creating a 3D homology model of KV7.2 in an open state suggesting that this residue plays a central role in the formation of a stable interface between the S1-S2 and the S5 segment helices. This could be the explanation why substitution of the conserved threonine in KV7.2 and KV7.3 channels destabilizes the open and favors the closed state of these channels.

Positive selection in octopus haemocyanin indicates functional links to temperature adaptation.

PubMed

Oellermann, Michael; Strugnell, Jan M; Lieb, Bernhard; Mark, Felix C

2015-07-05

Octopods have successfully colonised the world's oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods. Analysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence. This study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Crystal structure of LysK, an enzyme catalyzing the last step of lysine biosynthesis in Thermus thermophilus, in complex with lysine: Insight into the mechanism for recognition of the amino-group carrier protein, LysW.

PubMed

Fujita, Satomi; Cho, Su-Hee; Yoshida, Ayako; Hasebe, Fumihito; Tomita, Takeo; Kuzuyama, Tomohisa; Nishiyama, Makoto

2017-09-16

LysK is an M20 peptidase family enzyme that hydrolyzes the isopeptide bond between the carrier protein LysW and lysine in order to release lysine, which is the last step of lysine biosynthesis in Thermus thermophilus. In the present study, we determined the crystal structure of LysK in complex with lysine at a resolution of 2.4 Å. The α-amino group of the bound lysine was oriented toward the catalytic center, which was composed of the residues coordinating divalent metal ions for the hydrolysis of the isopeptide bond. An 11 Å-long path was observed from the active site binding lysine to the protein surface, which may be responsible for recognizing the C-terminal extension domain of LysW with the conserved EDWGE sequence. A positively-charged surface region was detected around the exit of the path, similar to other lysine biosynthetic enzymes using LysW as the carrier protein. Mutational studies of the surface residues provided a plausible model for the electrostatic interaction with LysW. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystal structure of the avilamycin resistance-conferring methyltransferase AviRa from Streptomyces viridochromogenes.

PubMed

Mosbacher, Tanja G; Bechthold, Andreas; Schulz, Georg E

2003-05-23

The emergence of antibiotic-resistant bacterial strains is a widespread problem in contemporary medical practice and drug design. It is therefore important to elucidate the underlying mechanism in each case. The methyltransferase AviRa from Streptomyces viridochromogenes mediates resistance to the antibiotic avilamycin, which is closely related to evernimicin, an oligosaccharide antibiotic that has been used in medical studies. The structure of AviRa was determined by X-ray diffraction at 1.5A resolution. Phases were obtained from one selenomethionine residue introduced by site-directed mutagenesis. The chain-fold is similar to that of most methyltransferases, although AviRa contains two additional helices as a specific feature. A putative-binding site for the cofactor S-adenosyl-L-methionine was derived from homologous structures. It agrees with the conserved pattern of interacting amino acid residues. AviRa methylates a specific guanine base within the peptidyltransferase loop of the 23S ribosomal RNA. Guided by the target, the enzyme was docked to the cognate ribosomal surface, where it fit well into a deep cleft without contacting any ribosomal protein. The two additional alpha-helices of AviRa filled a depression in the surface. Since the transferred methyl group of the cofactor is in a pocket beneath the enzyme surface, the targeted guanine base has to flip out for methylation.

Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics.

PubMed

Charpentier, S; Amiche, M; Mester, J; Vouille, V; Le Caer, J P; Nicolas, P; Delfour, A

1998-06-12

Analysis of antimicrobial activities that are present in the skin secretions of the South American frog Phyllomedusa bicolor revealed six polycationic (lysine-rich) and amphipathic alpha-helical peptides, 24-33 residues long, termed dermaseptins B1 to B6, respectively. Prepro-dermaseptins B all contain an almost identical signal peptide, which is followed by a conserved acidic propiece, a processing signal Lys-Arg, and a dermaseptin progenitor sequence. The 22-residue signal peptide plus the first 3 residues of the acidic propiece are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The 25-residue amino-terminal region of prepro-dermaseptins B shares 50% identity with the corresponding region of precursors for D-amino acid containing opioid peptides or for antimicrobial peptides originating from the skin of distantly related frog species. The remarkable similarity found between prepro-proteins that encode end products with strikingly different sequences, conformations, biological activities and modes of action suggests that the corresponding genes have evolved through dissemination of a conserved "secretory cassette" exon.

Soil Carbon Changes in Transitional Grain Crop Production Systems in South Dakota

NASA Astrophysics Data System (ADS)

Woodard, H. J.

2004-12-01

Corn-C (Zea Mays L.), soybean-S (Glycine max L.) and spring wheat-W (Triticum aestivum L.) crops were seeded as a component of either a C-S, S-W, or C-S-W crop rotation on silt-loam textured soils ranging from 3.0-5.0% organic matter. Conservation tillage(chisel plow-field cultivator) was applied to half of the plots. The other plots were direct seeded as a no-till (zero-tillage) treatment. Grain yield and surface crop residues were weighed from each treatment plot. Crop residue (stover and straw) was removed from half of the plots. After four years, soil samples were removed at various increments of depth and soil organic carbon (C) and nitrogen (N) was measured. The ranking of crop residue weights occurred by the order corn>>soybean>wheat. Surface residue accumulation was also greatest with residue treatments that were returned to the plots, those rotations in which maize was a component, and those without tillage. Mean soil organic carbon levels in the 0-7.5cm depth decreased from 3.41% to 3.19% (- 0.22%) with conventional tillage (chisel plow/field cultivator) as compared to a decrease from 3.19% to 3.05% (-0.14%) in plots without tillage over a four year period. Organic carbon in the 0-7.5cm depth decreased from 3.21% to 3.01% (- 0.20%) after residue removed as compared to a decrease from 3.39% to 3.23% (-0.17%) in plots without tillage applied after four years. The soil C:N ratio (0-7.5cm) decreased from 10.63 to 10.37 (-0.26 (unitless)) in the tilled plots over a four-year period. Soil C:N ratio at the 0-7.5cm depth decreased from 10.72 to 10.04 (-0.68) in the no-till plots over a four year period. Differences in the soil C:N ratio comparing residue removed and residue returned were similar (-0.51 vs. -0.43 respectively). These soils are highly buffered for organic carbon changes. Many cropping cycles are required to determine how soil carbon storage is significantly impacted by production systems.

Structure and stability of the ankyrin domain of the Drosophila Notch receptor.

PubMed

Zweifel, Mark E; Leahy, Daniel J; Hughson, Frederick M; Barrick, Doug

2003-11-01

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.

Novel microfabrication stage allowing for one-photon and multi-photon light assisted molecular immobilization and for multi-photon microscope

NASA Astrophysics Data System (ADS)

Gonçalves, Odete; Snider, Scott; Zadoyan, Ruben; Nguyen, Quoc-Thang; Vorum, Henrik; Petersen, Steffen B.; Neves-Petersen, Maria Teresa

2017-02-01

Light Assisted Molecular Immobilization (LAMI) results in spatially oriented and localized covalent coupling of biomolecules onto thiol reactive surfaces. LAMI is possible due to the conserved spatial proximity between aromatic residues and disulfide bridges in proteins. When aromatic residues are excited with UV light (275-295nm), disulphide bridges are disrupted and the formed thiol groups covalently bind to surfaces. Immobilization hereby reported is achieved in a microfabrication stage coupled to a fs-laser, through one- or multi-photon excitation. The fundamental 840nm output is tripled to 280nm and focused onto the sample, leading to one-photon excitation and molecular immobilization. The sample rests on a xyz-stage with micrometer step resolution and is illuminated according to a pattern uploaded to the software controlling the stage and the shutter. Molecules are immobilized according to such pattern, with micrometer spatial resolution. Spatial masks inserted in the light path lead to light diffraction patterns used to immobilize biomolecules with submicrometer spatial resolution. Light diffraction patterns are imaged by an inbuilt microscope. Two-photon microscopy and imaging of the fluorescent microbeads is shown. Immobilization of proteins, e.g. C-reactive protein, and of an engineered molecular beacon has been successfully achieved. The beacon was coupled to a peptide containing a disulfide bridge neighboring a tryptophan residue, being this way possible to immobilize the beacon on a surface using one-photon LAMI. This technology is being implemented in the creation of point-of-care biosensors aiming at the detection of cancer and cardiovascular disease markers.

Diversified Structural Basis of a Conserved Molecular Mechanism for pH-Dependent Dimerization in Spider Silk N-Terminal Domains.

PubMed

Otikovs, Martins; Chen, Gefei; Nordling, Kerstin; Landreh, Michael; Meng, Qing; Jörnvall, Hans; Kronqvist, Nina; Rising, Anna; Johansson, Jan; Jaudzems, Kristaps

2015-08-17

Conversion of spider silk proteins from soluble dope to insoluble fibers involves pH-dependent dimerization of the N-terminal domain (NT). This conversion is tightly regulated to prevent premature precipitation and enable rapid silk formation at the end of the duct. Three glutamic acid residues that mediate this process in the NT from Euprosthenops australis major ampullate spidroin 1 are well conserved among spidroins. However, NTs of minor ampullate spidroins from several species, including Araneus ventricosus ((Av)MiSp NT), lack one of the glutamic acids. Here we investigate the pH-dependent structural changes of (Av)MiSp NT, revealing that it uses the same mechanism but involves a non-conserved glutamic acid residue instead. Homology modeling of the structures of other MiSp NTs suggests that these harbor different compensatory residues. This indicates that, despite sequence variations, the molecular mechanism underlying pH-dependent dimerization of NT is conserved among different silk types. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequence, structure and function relationships in flaviviruses as assessed by evolutive aspects of its conserved non-structural protein domains.

PubMed

da Fonseca, Néli José; Lima Afonso, Marcelo Querino; Pedersolli, Natan Gonçalves; de Oliveira, Lucas Carrijo; Andrade, Dhiego Souto; Bleicher, Lucas

2017-10-28

Flaviviruses are responsible for serious diseases such as dengue, yellow fever, and zika fever. Their genomes encode a polyprotein which, after cleavage, results in three structural and seven non-structural proteins. Homologous proteins can be studied by conservation and coevolution analysis as detected in multiple sequence alignments, usually reporting positions which are strictly necessary for the structure and/or function of all members in a protein family or which are involved in a specific sub-class feature requiring the coevolution of residue sets. This study provides a complete conservation and coevolution analysis on all flaviviruses non-structural proteins, with results mapped on all well-annotated available sequences. A literature review on the residues found in the analysis enabled us to compile available information on their roles and distribution among different flaviviruses. Also, we provide the mapping of conserved and coevolved residues for all sequences currently in SwissProt as a supplementary material, so that particularities in different viruses can be easily analyzed. Copyright © 2017 Elsevier Inc. All rights reserved.

A Conserved Asparagine Residue in Transmembrane Segment 1 (TM1) of Serotonin Transporter Dictates Chloride-coupled Neurotransmitter Transport*

PubMed Central

Henry, L. Keith; Iwamoto, Hideki; Field, Julie R.; Kaufmann, Kristian; Dawson, Eric S.; Jacobs, Miriam T.; Adams, Chelsea; Felts, Bruce; Zdravkovic, Igor; Armstrong, Vanessa; Combs, Steven; Solis, Ernesto; Rudnick, Gary; Noskov, Sergei Y.; DeFelice, Louis J.; Meiler, Jens; Blakely, Randy D.

2011-01-01

Na+- and Cl−-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl− coordination of human serotonin transport have been identified, the role of Cl− in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl− binding to concentrative neurotransmitter uptake. PMID:21730057

Identification of kinetically hot residues in proteins.

PubMed Central

Demirel, M. C.; Atilgan, A. R.; Jernigan, R. L.; Erman, B.; Bahar, I.

1998-01-01

A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed. PMID:9865946

[Three regions of Rpb10 mini-subunit of nuclear RNA polymerases are strictly conserved in all eukaryotes].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N

1996-12-01

The rpb10+ cDNA from the fission yeast Schizosaccharomyces pombe was cloned using two independent approaches (PCR and genetic suppression). The cloned cDNA encoded the Rpb10 subunit common for all three RNA polymerases. Comparison of the deduced amino acid sequence of the Sz. pombe Rbp10 subunit (71 amino acid residues) with those of the homologous subunits of RNA polymerases I, II, and III from Saccharomyces cerevisiae and Home sapiens revealed that heptapeptides RCFT/SCGK (residues 6-12), RYCCRRM (residues 43-49), and HVDLIEK (residues 53-59) were evolutionarily the most conserved structural motifs of these subunits. It is shown that the Rbp10 subunit from Sz. pombe can substitute its homolog (ABC10 beta) in the baker's yeast S. cerevisiae.

1H, 13C and 15N assignment of the C-terminal domain of GNA2132 from Neisseria meningitidis

PubMed Central

Esposito, Veronica; Musi, Valeria; Veggi, Daniele; Pizza, Mariagrazia

2010-01-01

GNA2132 (Genome-derived Neisseria Antigen 2132) is a surface-exposed lipoprotein discovered by reverse vaccinology and expressed by genetically diverse Neisseria meningitidis strains (Pizza et al. 2000). The protein induces bactericidal antibodies against most strains of Meningococccus and has been included in a multivalent recombinant vaccine against N. meningitidis serogroup B. Structure determination of GNA2132 is important for understanding the antigenic properties of the protein in view of increased efficiency vaccine development. We report practically complete 1H, 13C and 15N assignment of the detectable spectrum of a highly conserved C-terminal region of GNA2132 (residues 245–427) in micellar solution, a medium used to improve the spectral quality. The first 32 residues of our construct up to residue 277 were not visible in the spectrum, presumably because of line broadening due to solvent and/or conformational exchange. Secondary structure predictions based on chemical shift information indicate the presence of an all β-protein with eight β strands. PMID:20300890

Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport

PubMed Central

Sherman, David J.; Lazarus, Michael B.; Murphy, Lea; Liu, Charles; Walker, Suzanne; Ruiz, Natividad; Kahne, Daniel

2014-01-01

The cell surface of Gram-negative bacteria contains lipopolysaccharides (LPS), which provide a barrier against the entry of many antibiotics. LPS assembly involves a multiprotein LPS transport (Lpt) complex that spans from the cytoplasm to the outer membrane. In this complex, an unusual ATP-binding cassette transporter is thought to power the extraction of LPS from the outer leaflet of the cytoplasmic membrane and its transport across the cell envelope. We introduce changes into the nucleotide-binding domain, LptB, that inactivate transporter function in vivo. We characterize these residues using biochemical experiments combined with high-resolution crystal structures of LptB pre- and post-ATP hydrolysis and suggest a role for an active site residue in phosphate exit. We also identify a conserved residue that is not required for ATPase activity but is essential for interaction with the transmembrane components. Our studies establish the essentiality of ATP hydrolysis by LptB to power LPS transport in cells and suggest strategies to inhibit transporter function away from the LptB active site. PMID:24639492

Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeast Glaciozyma antarctica PI12.

PubMed

Jaafar, Nardiah Rizwana; Littler, Dene; Beddoe, Travis; Rossjohn, Jamie; Illias, Rosli Md; Mahadi, Nor Muhammad; Mackeen, Mukram Mohamed; Murad, Abdul Munir Abdul; Abu Bakar, Farah Diba

2016-11-01

Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.

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.

Functional region prediction with a set of appropriate homologous sequences-an index for sequence selection by integrating structure and sequence information with spatial statistics

PubMed Central

2012-01-01

Background The detection of conserved residue clusters on a protein structure is one of the effective strategies for the prediction of functional protein regions. Various methods, such as Evolutionary Trace, have been developed based on this strategy. In such approaches, the conserved residues are identified through comparisons of homologous amino acid sequences. Therefore, the selection of homologous sequences is a critical step. It is empirically known that a certain degree of sequence divergence in the set of homologous sequences is required for the identification of conserved residues. However, the development of a method to select homologous sequences appropriate for the identification of conserved residues has not been sufficiently addressed. An objective and general method to select appropriate homologous sequences is desired for the efficient prediction of functional regions. Results We have developed a novel index to select the sequences appropriate for the identification of conserved residues, and implemented the index within our method to predict the functional regions of a protein. The implementation of the index improved the performance of the functional region prediction. The index represents the degree of conserved residue clustering on the tertiary structure of the protein. For this purpose, the structure and sequence information were integrated within the index by the application of spatial statistics. Spatial statistics is a field of statistics in which not only the attributes but also the geometrical coordinates of the data are considered simultaneously. Higher degrees of clustering generate larger index scores. We adopted the set of homologous sequences with the highest index score, under the assumption that the best prediction accuracy is obtained when the degree of clustering is the maximum. The set of sequences selected by the index led to higher functional region prediction performance than the sets of sequences selected by other sequence-based methods. Conclusions Appropriate homologous sequences are selected automatically and objectively by the index. Such sequence selection improved the performance of functional region prediction. As far as we know, this is the first approach in which spatial statistics have been applied to protein analyses. Such integration of structure and sequence information would be useful for other bioinformatics problems. PMID:22643026

A Conserved Aspartate Residue Located at the Extracellular End of the Binding Pocket Controls Cation Interactions in Brain Glutamate Transporters*

PubMed Central

Rosental, Noa; Gameiro, Armanda; Grewer, Christof; Kanner, Baruch I.

2011-01-01

In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na+ ions, followed by countertransport of K+. Recent studies, based on several crystal structures of the archeal homologue GltPh, indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na+ binding, but functional and computational studies suggest some candidate sites. In the GltPh structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of GltPh. Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na+ and K+, respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters. PMID:21984827

A conserved aspartate residue located at the extracellular end of the binding pocket controls cation interactions in brain glutamate transporters.

PubMed

Rosental, Noa; Gameiro, Armanda; Grewer, Christof; Kanner, Baruch I

2011-12-02

In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na(+) ions, followed by countertransport of K(+). Recent studies, based on several crystal structures of the archeal homologue Glt(Ph), indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na(+) binding, but functional and computational studies suggest some candidate sites. In the Glt(Ph) structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt(Ph). Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na(+) and K(+), respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.

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)

Peanut residue carbon and nitrogen mineralization under simulated conventional and conservation tillage

USDA-ARS?s Scientific Manuscript database

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 during decomposition. Cooperative Extension currently recommends 22-67 kg N ha-1 credit to subsequent crops following peanut (Ar...

A Bioinformatics Approach to the Structure, Function, and Evolution of the Nucleoprotein of the Order Mononegavirales

PubMed Central

Cleveland, Sean B.; Davies, John; McClure, Marcella A.

2011-01-01

The goal of this Bioinformatic study is to investigate sequence conservation in relation to evolutionary function/structure of the nucleoprotein of the order Mononegavirales. In the combined analysis of 63 representative nucleoprotein (N) sequences from four viral families (Bornaviridae, Filoviridae, Rhabdoviridae, and Paramyxoviridae) we predict the regions of protein disorder, intra-residue contact and co-evolving residues. Correlations between location and conservation of predicted regions illustrate a strong division between families while high- lighting conservation within individual families. These results suggest the conserved regions among the nucleoproteins, specifically within Rhabdoviridae and Paramyxoviradae, but also generally among all members of the order, reflect an evolutionary advantage in maintaining these sites for the viral nucleoprotein as part of the transcription/replication machinery. Results indicate conservation for disorder in the C-terminus region of the representative proteins that is important for interacting with the phosphoprotein and the large subunit polymerase during transcription and replication. Additionally, the C-terminus region of the protein preceding the disordered region, is predicted to be important for interacting with the encapsidated genome. Portions of the N-terminus are responsible for N∶N stability and interactions identified by the presence or lack of co-evolving intra-protein contact predictions. The validation of these prediction results by current structural information illustrates the benefits of the Disorder, Intra-residue contact and Compensatory mutation Correlator (DisICC) pipeline as a method for quickly characterizing proteins and providing the most likely residues and regions necessary to target for disruption in viruses that have little structural information available. PMID:21559282

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

Comparative sequence analysis of acid sensitive/resistance proteins in Escherichia coli and Shigella flexneri

PubMed Central

Manikandan, Selvaraj; Balaji, Seetharaaman; Kumar, Anil; Kumar, Rita

2007-01-01

The molecular basis for the survival of bacteria under extreme conditions in which growth is inhibited is a question of great current interest. A preliminary study was carried out to determine residue pattern conservation among the antiporters of enteric bacteria, responsible for extreme acid sensitivity especially in Escherichia coli and Shigella flexneri. Here we found the molecular evidence that proved the relationship between E. coli and S. flexneri. Multiple sequence alignment of the gadC coded acid sensitive antiporter showed many conserved residue patterns at regular intervals at the N-terminal region. It was observed that as the alignment approaches towards the C-terminal, the number of conserved residues decreases, indicating that the N-terminal region of this protein has much active role when compared to the carboxyl terminal. The motif, FHLVFFLLLGG, is well conserved within the entire gadC coded protein at the amino terminal. The motif is also partially conserved among other antiporters (which are not coded by gadC) but involved in acid sensitive/resistance mechanism. Phylogenetic cluster analysis proves the relationship of Escherichia coli and Shigella flexneri. The gadC coded proteins are converged as a clade and diverged from other antiporters belongs to the amino acid-polyamine-organocation (APC) superfamily. PMID:21670792

Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry.

PubMed

Rhein, Bethany A; Brouillette, Rachel B; Schaack, Grace A; Chiorini, John A; Maury, Wendy

2016-07-01

Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion-TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions. With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses, including EBOV. TIM family member TIM-4 is expressed on macrophages and dendritic cells, which are early cellular targets during EBOV infection. Here, we performed a mutagenesis screening of the IgV domain of murine and human TIM-4 to identify residues that are critical for EBOV entry. Surprisingly, we identified more human than murine TIM-4 IgV domain residues that are required for EBOV entry. Defining the TIM IgV residues needed for EBOV entry clarifies the virus-receptor interactions and paves the way for the development of novel therapeutics targeting virus binding to this cell surface receptor. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry

PubMed Central

Rhein, Bethany A.; Brouillette, Rachel B.; Schaack, Grace A.; Chiorini, John A.

2016-01-01

ABSTRACT Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion–TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions. IMPORTANCE With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses, including EBOV. TIM family member TIM-4 is expressed on macrophages and dendritic cells, which are early cellular targets during EBOV infection. Here, we performed a mutagenesis screening of the IgV domain of murine and human TIM-4 to identify residues that are critical for EBOV entry. Surprisingly, we identified more human than murine TIM-4 IgV domain residues that are required for EBOV entry. Defining the TIM IgV residues needed for EBOV entry clarifies the virus-receptor interactions and paves the way for the development of novel therapeutics targeting virus binding to this cell surface receptor. PMID:27122575

Pulmonary stenosis and pulmonary regurgitation: both ends of the spectrum in residual hemodynamic impairment after tetralogy of Fallot repair.

PubMed

Yoo, Byung Won; Park, Han Ki

2013-06-01

Repair of tetralogy of Fallot (TOF) has shown excellent outcomes. However it leaves varying degrees of residual hemodynamic impairment, with severe pulmonary stenosis (PS) and free pulmonary regurgitation (PR) at both ends of the spectrum. Since the 1980s, studies evaluating late outcomes after TOF repair revealed the adverse impacts of residual chronic PR on RV volume and function; thus, a turnaround of operational strategies has occurred from aggressive RV outflow tract (RVOT) reconstruction for complete relief of RVOT obstruction to conservative RVOT reconstruction for limiting PR. This transformation has raised the question of how much residual PS after conservative RVOT reconstruction is acceptable. Besides, as pulmonary valve replacement (PVR) increases in patients with RV deterioration from residual PR, there is concern regarding when it should be performed. Regarding residual PS, several studies revealed that PS in addition to PR was associated with less PR and a small RV volume. This suggests that PS combined with PR makes RV diastolic property to protect against dilatation through RV hypertrophy and supports conservative RVOT enlargement despite residual PS. Also, several studies have revealed the pre-PVR threshold of RV parameters for the normalization of RV volume and function after PVR, and based on these results, the indications for PVR have been revised. Although there is no established strategy, better understanding of RV mechanics, development of new surgical and interventional techniques, and evidence for the effect of PVR on RV reverse remodeling and its late outcome will aid us to optimize the management of TOF.

Herbicide and cover crop residue integration in conservation tillage tomato

USDA-ARS?s Scientific Manuscript database

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...

A Proteome-wide Domain-centric Perspective on Protein Phosphorylation *

PubMed Central

Palmeri, Antonio; Ausiello, Gabriele; Ferrè, Fabrizio; Helmer-Citterich, Manuela; Gherardini, Pier Federico

2014-01-01

Phosphorylation is a widespread post-translational modification that modulates the function of a large number of proteins. Here we show that a significant proportion of all the domains in the human proteome is significantly enriched or depleted in phosphorylation events. A substantial improvement in phosphosites prediction is achieved by leveraging this observation, which has not been tapped by existing methods. Phosphorylation sites are often not shared between multiple occurrences of the same domain in the proteome, even when the phosphoacceptor residue is conserved. This is partly because of different functional constraints acting on the same domain in different protein contexts. Moreover, by augmenting domain alignments with structural information, we were able to provide direct evidence that phosphosites in protein-protein interfaces need not be positionally conserved, likely because they can modulate interactions simply by sitting in the same general surface area. PMID:24830415

Structural analysis of a functional DIAP1 fragment bound to grim and hid peptides.

PubMed

Wu, J W; Cocina, A E; Chai, J; Hay, B A; Shi, Y

2001-07-01

The inhibitor of apoptosis protein DIAP1 suppresses apoptosis in Drosophila, with the second BIR domain (BIR2) playing an important role. Three proteins, Hid, Grim, and Reaper, promote apoptosis, in part by binding to DIAP1 through their conserved N-terminal sequences. The crystal structures of DIAP1-BIR2 by itself and in complex with the N-terminal peptides from Hid and Grim reveal that these peptides bind a surface groove on DIAP1, with the first four amino acids mimicking the binding of the Smac tetrapeptide to XIAP. The next 3 residues also contribute to binding through hydrophobic interactions. Interestingly, peptide binding induces the formation of an additional alpha helix in DIAP1. Our study reveals the structural conservation and diversity necessary for the binding of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins.

Atomic basis for therapeutic activation of neuronal potassium channels

NASA Astrophysics Data System (ADS)

Kim, Robin Y.; Yau, Michael C.; Galpin, Jason D.; Seebohm, Guiscard; Ahern, Christopher A.; Pless, Stephan A.; Kurata, Harley T.

2015-09-01

Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2-5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.

Roles of the conserved cytoplasmic region and non-conserved carboxy-terminal region of SecE in Escherichia coli protein translocase.

PubMed

Kontinen, V P; Yamanaka, M; Nishiyama, K; Tokuda, H

1996-06-01

SecE, an essential membrane component of the Escherichia coli protein translocase, consists of 127 amino acid residues. Only a part of the second putative cytoplasmic region comprising some 13 residues is essential for the SecE function as long as the proper topological arrangement is retained. The Trp84 and Pro85 residues of this region are conserved in all eubacterial SecE homologues. The conservation of positively charged residues corresponding to Arg80 and Lys81 is also substantial. We deleted or replaced these residues to assess their roles in the SecE function. Deletion of the Arg80-Lys81 dipeptide did not abolish the SecE function whereas that of Trp84 or Pro85 caused a loss of the function. Strikingly, however, replacement of Pro85 with either Gly, Ser, or Ala, and that of Trp84 with Lys did not abolish the SecE function. These results indicate that the strong conservation of these residues does not reflect their obligatory requirement for the SecE function. A chimeric SecE possessing the cytoplasmic region of the E. coli SecE and the following region of the Bacillus subtilis SecE was able to form the translocation machinery together with SecA, SecY, and SecG. Although a Leu to Arg mutation at position 108 has been thought to cause a loss of signal recognition fidelity and thereby suppress a signal sequence defect, the same mutation at position 111 caused a complete loss of the function. The levels of SecY and SecG in the secEcsE501 mutant, which expresses SecE at a decreased level and is sensitive to low temperature, increased upon the expression of functional SecE derivatives, irrespective of the site of mutation, suggesting that the levels of SecY and SecG are co-operatively determined by the level of functional, but not non-functional, SecE. Based on these results, the SecE function in the translocase is discussed.

Modeling coding-sequence evolution within the context of residue solvent accessibility.

PubMed

Scherrer, Michael P; Meyer, Austin G; Wilke, Claus O

2012-09-12

Protein structure mediates site-specific patterns of sequence divergence. In particular, residues in the core of a protein (solvent-inaccessible residues) tend to be more evolutionarily conserved than residues on the surface (solvent-accessible residues). Here, we present a model of sequence evolution that explicitly accounts for the relative solvent accessibility of each residue in a protein. Our model is a variant of the Goldman-Yang 1994 (GY94) model in which all model parameters can be functions of the relative solvent accessibility (RSA) of a residue. We apply this model to a data set comprised of nearly 600 yeast genes, and find that an evolutionary-rate ratio ω that varies linearly with RSA provides a better model fit than an RSA-independent ω or an ω that is estimated separately in individual RSA bins. We further show that the branch length t and the transition-transverion ratio κ also vary with RSA. The RSA-dependent GY94 model performs better than an RSA-dependent Muse-Gaut 1994 (MG94) model in which the synonymous and non-synonymous rates individually are linear functions of RSA. Finally, protein core size affects the slope of the linear relationship between ω and RSA, and gene expression level affects both the intercept and the slope. Structure-aware models of sequence evolution provide a significantly better fit than traditional models that neglect structure. The linear relationship between ω and RSA implies that genes are better characterized by their ω slope and intercept than by just their mean ω.

Conformational Occlusion of Blockade Antibody Epitopes, a Novel Mechanism of GII.4 Human Norovirus Immune Evasion.

PubMed

Lindesmith, Lisa C; Mallory, Michael L; Debbink, Kari; Donaldson, Eric F; Brewer-Jensen, Paul D; Swann, Excel W; Sheahan, Timothy P; Graham, Rachel L; Beltramello, Martina; Corti, Davide; Lanzavecchia, Antonio; Baric, Ralph S

2018-01-01

Extensive antigenic diversity within the GII.4 genotype of human norovirus is a major driver of pandemic emergence and a significant obstacle to development of cross-protective immunity after natural infection and vaccination. However, human and mouse monoclonal antibody studies indicate that, although rare, antibodies to conserved GII.4 blockade epitopes are generated. The mechanisms by which these epitopes evade immune surveillance are uncertain. Here, we developed a new approach for identifying conserved GII.4 norovirus epitopes. Utilizing a unique set of virus-like particles (VLPs) representing the in vivo -evolved sequence diversity within an immunocompromised person, we identify key residues within epitope F, a conserved GII.4 blockade antibody epitope. The residues critical for antibody binding are proximal to evolving blockade epitope E. Like epitope F, antibody blockade of epitope E was temperature sensitive, indicating that particle conformation regulates antibody access not only to the conserved GII.4 blockade epitope F but also to the evolving epitope E. These data highlight novel GII.4 mechanisms to protect blockade antibody epitopes, map essential residues of a GII.4 conserved epitope, and expand our understanding of how viral particle dynamics may drive antigenicity and antibody-mediated protection by effectively shielding blockade epitopes. Our data support the notion that GII.4 particle breathing may well represent a major mechanism of humoral immune evasion supporting cyclic pandemic virus persistence and spread in human populations. IMPORTANCE In this study, we use norovirus virus-like particles to identify key residues of a conserved GII.4 blockade antibody epitope. Further, we identify an additional GII.4 blockade antibody epitope to be occluded, with antibody access governed by temperature and particle dynamics. These findings provide additional support for particle conformation-based presentation of binding residues mediated by a particle "breathing core." Together, these data suggest that limiting antibody access to blockade antibody epitopes may be a frequent mechanism of immune evasion for GII.4 human noroviruses. Mapping blockade antibody epitopes, the interaction between adjacent epitopes on the particle, and the breathing core that mediates antibody access to epitopes provides greater mechanistic understanding of epitope camouflage strategies utilized by human viral pathogens to evade immunity.

Structure of the Minor Pseudopilin EpsH From the Type 2 Secretion System of Vibrio Cholerae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanez, M.E.; Korotkov, K.V.; Abendroth, J.

2009-05-28

Many Gram-negative bacteria use the multi-protein type II secretion system (T2SS) to selectively translocate virulence factors from the periplasmic space into the extracellular environment. In Vibrio cholerae the T2SS is called the extracellular protein secretion (Eps) system, which translocates cholera toxin and several enzymes in their folded state across the outer membrane. Five proteins of the T2SS, the pseudopilins, are thought to assemble into a pseudopilus, which may control the outer membrane pore EpsD, and participate in the active export of proteins in a 'piston-like' manner. We report here the 2.0 {angstrom} resolution crystal structure of an N-terminally truncated variantmore » of EpsH, a minor pseudopilin from Vibrio cholerae. While EpsH maintains an N-terminal {alpha}-helix and C-terminal {beta}-sheet consistent with the type 4a pilin fold, structural comparisons reveal major differences between the minor pseudopilin EpsH and the major pseudopilin GspG from Klebsiella oxytoca: EpsH contains a large {beta}-sheet in the variable domain, where GspG contains an {alpha}-helix. Most importantly, EpsH contains at its surface a hydrophobic crevice between its variable and conserved {beta}-sheets, wherein a majority of the conserved residues within the EpsH family are clustered. In a tentative model of a T2SS pseudopilus with EpsH at its tip, the conserved crevice faces away from the helix axis. This conserved surface region may be critical for interacting with other proteins from the T2SS machinery.« less

Identification of a divalent metal cation binding site in herpes simplex virus 1 (HSV-1) ICP8 required for HSV replication.

PubMed

Bryant, Kevin F; Yan, Zhipeng; Dreyfus, David H; Knipe, David M

2012-06-01

Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro. Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

PubMed

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

Prediction of the interaction site on the surface of an isolated protein structure by analysis of side chain energy scores.

PubMed

Liang, Shide; Zhang, Jian; Zhang, Shicui; Guo, Huarong

2004-11-15

We show that residues at the interfaces of protein-protein complexes have higher side-chain energy than other surface residues. Eight different sets of protein complexes were analyzed. For each protein pair, the complex structure was used to identify the interface residues in the unbound monomer structures. Side-chain energy was calculated for each surface residue in the unbound monomer using our previously developed scoring function.1 The mean energy was calculated for the interface residues and the other surface residues. In 15 of the 16 monomers, the mean energy of the interface residues was higher than that of other surface residues. By decomposing the scoring function, we found that the energy term of the buried surface area of non-hydrogen-bonded hydrophilic atoms is the most important factor contributing to the high energy of the interface regions. In spite of lacking hydrophilic residues, the interface regions were found to be rich in buried non-hydrogen-bonded hydrophilic atoms. Although the calculation results could be affected by the inaccuracy of the scoring function, patch analysis of side-chain energy on the surface of an isolated protein may be helpful in identifying the possible protein-protein interface. A patch was defined as 20 residues surrounding the central residue on the protein surface, and patch energy was calculated as the mean value of the side-chain energy of all residues in the patch. In 12 of the studied monomers, the patch with the highest energy overlaps with the observed interface. The results are more remarkable when only three residues with the highest energy in a patch are averaged to derive the patch energy. All three highest-energy residues of the top energy patch belong to interfacial residues in four of the eight small protomers. We also found that the residue with the highest energy score on the surface of a small protomer is very possibly the key interaction residue. (c) 2004 Wiley-Liss, Inc.

77 FR 39895 - New Analytic Methods and Sampling Procedures for the United States National Residue Program for...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-06

... Analytic Methods and Sampling Procedures for the United States National Residue Program for Meat, Poultry... implementing several multi-residue methods for analyzing samples of meat, poultry, and egg products for animal.... These modern, high-efficiency methods will conserve resources and provide useful and reliable results...

High Residue Winter Cover Crops Deplete Winter Annual Weed Seed Across a Landscape in a Long-Term Tillage Study

USDA-ARS?s Scientific Manuscript database

High residue conservation agriculture systems have the potential to maximize environmental benefits achieved when practicing reduced tillage. A greenhouse study was conducted in 2006 through 2008 to determine the effects of cover crop residue on weed seed density within the soil seedbank under varyi...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Tarini Shankar; Chaitanya, S. Krishna; Sankararamakrishnan, Ramasubbu, E-mail: rsankar@iitk.ac.in

New classes of helix–helix interactions in protein structures are reported in which interactions only occur at the terminal regions or between the terminal region of one helix and the middle region of another helix. Helix–helix interactions are important for the structure, stability and function of α-helical proteins. Helices that either cross in the middle or show extensive contacts between each other, such as coiled coils, have been investigated in previous studies. Interactions between two helices can also occur only at the terminal regions or between the terminal region of one helix and the middle region of another helix. Examples ofmore » such helix pairs are found in aquaporin, H{sup +}/Cl{sup −} transporter and Bcl-2 proteins. The frequency of the occurrence of such ‘end-to-end’ (EE) and ‘end-to-middle’ (EM) helix pairs in protein structures is not known. Questions regarding the residue preferences in the interface and the mode of interhelical interactions in such helix pairs also remain unanswered. In this study, high-resolution structures of all-α proteins from the PDB have been systematically analyzed and the helix pairs that interact only in EE or EM fashion have been extracted. EE and EM helix pairs have been categorized into five classes (N–N, N–C, C–C, N–MID and C–MID) depending on the region of interaction. Nearly 13% of 5725 helix pairs belonged to one of the five classes. Analysis of single-residue propensities indicated that hydrophobic and polar residues prefer to occur in the C-terminal and N-terminal regions, respectively. Hydrophobic C-terminal interacting residues and polar N-terminal interacting residues are also highly conserved. A strong correlation exists between some of the residue properties (surface area/volume and length of side chains) and their preferences for occurring in the interface of EE and EM helix pairs. In contrast to interacting non-EE/EM helix pairs, helices in EE and EM pairs are farther apart. In these helix pairs, residues with large surface area/volume and longer side chains are preferred in the interfacial region.« less

An insight to the dynamics of conserved water molecular triad in IMPDH II (human): recognition of cofactor and substrate to catalytic Arg 322.

PubMed

Bairagya, Hridoy R; Mukhopadhyay, Bishnu P; Sekar, K

2009-10-01

Inosine 5' monophosphate dehydrogenase (IMPDH II) is a key enzyme involved in the de novo biosynthesis pathway of purine nucleotides and is also considered to be an excellent target for cancer inhibitor design. The conserve R 322 residue (in human) is thought to play some role in the recognition of inhibitor and cofactor through the catalytic D 364 and N 303. The 15 ns simulation and the water dynamics of the three different PDB structures (1B3O, 1NF7, and 1NFB) of human IMPDH by CHARMM force field have clearly indicated the involvement of three conserved water molecules (W(L), W(M), and W(C)) in the recognition of catalytic residues (R 322, D 364, and N 303) to inhibitor and cofactor. Both the guanidine nitrogen atoms (NH1 and NH 2) of the R 322 have anchored the di- and mono-nucleotide (cofactor and inhibitor) binding domains via the conserved W(C) and W(L) water molecules. Another conserved water molecule WM seems to bridge the two domains including the R 322 and also the W(C) and W(L) through seven centers H-bonding coordination. The conserved water molecular triad (W(C)-W(M)-W(L)) in the protein complex may thought to play some important role in the recognition of inhibitor and cofactor to the protein through R 322 residue.

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

Molecular Evolution of Vertebrate Neurotrophins: Co-Option of the Highly Conserved Nerve Growth Factor Gene into the Advanced Snake Venom Arsenalf

PubMed Central

Sunagar, Kartik; Fry, Bryan Grieg; Jackson, Timothy N. W.; Casewell, Nicholas R.; Undheim, Eivind A. B.; Vidal, Nicolas; Ali, Syed A.; King, Glenn F.; Vasudevan, Karthikeyan; Vasconcelos, Vitor; Antunes, Agostinho

2013-01-01

Neurotrophins are a diverse class of structurally related proteins, essential for neuronal development, survival, plasticity and regeneration. They are characterized by major family members, such as the nerve growth factors (NGF), brain-derived neurotrophic factors (BDNF) and neurotrophin-3 (NT-3), which have been demonstrated here to lack coding sequence variations and follow the regime of negative selection, highlighting their extremely important conserved role in vertebrate homeostasis. However, in stark contrast, venom NGF secreted as part of the chemical arsenal of the venomous advanced snake family Elapidae (and to a lesser extent Viperidae) have characteristics consistent with the typical accelerated molecular evolution of venom components. This includes a rapid rate of diversification under the significant influence of positive-selection, with the majority of positively-selected sites found in the secreted β-polypeptide chain (74%) and on the molecular surface of the protein (92%), while the core structural and functional residues remain highly constrained. Such focal mutagenesis generates active residues on the toxin molecular surface, which are capable of interacting with novel biological targets in prey to induce a myriad of pharmacological effects. We propose that caenophidian NGFs could participate in prey-envenoming by causing a massive release of chemical mediators from mast cells to mount inflammatory reactions and increase vascular permeability, thereby aiding the spread of other toxins and/or by acting as proapoptotic factors. Despite their presence in reptilian venom having been known for over 60 years, this is the first evidence that venom-secreted NGF follows the molecular evolutionary pattern of other venom components, and thus likely participates in prey-envenomation. PMID:24312363

Molecular evolution of vertebrate neurotrophins: co-option of the highly conserved nerve growth factor gene into the advanced snake venom arsenalf.

PubMed

Sunagar, Kartik; Fry, Bryan Grieg; Jackson, Timothy N W; Casewell, Nicholas R; Undheim, Eivind A B; Vidal, Nicolas; Ali, Syed A; King, Glenn F; Vasudevan, Karthikeyan; Vasconcelos, Vitor; Antunes, Agostinho

2013-01-01

Neurotrophins are a diverse class of structurally related proteins, essential for neuronal development, survival, plasticity and regeneration. They are characterized by major family members, such as the nerve growth factors (NGF), brain-derived neurotrophic factors (BDNF) and neurotrophin-3 (NT-3), which have been demonstrated here to lack coding sequence variations and follow the regime of negative selection, highlighting their extremely important conserved role in vertebrate homeostasis. However, in stark contrast, venom NGF secreted as part of the chemical arsenal of the venomous advanced snake family Elapidae (and to a lesser extent Viperidae) have characteristics consistent with the typical accelerated molecular evolution of venom components. This includes a rapid rate of diversification under the significant influence of positive-selection, with the majority of positively-selected sites found in the secreted β-polypeptide chain (74%) and on the molecular surface of the protein (92%), while the core structural and functional residues remain highly constrained. Such focal mutagenesis generates active residues on the toxin molecular surface, which are capable of interacting with novel biological targets in prey to induce a myriad of pharmacological effects. We propose that caenophidian NGFs could participate in prey-envenoming by causing a massive release of chemical mediators from mast cells to mount inflammatory reactions and increase vascular permeability, thereby aiding the spread of other toxins and/or by acting as proapoptotic factors. Despite their presence in reptilian venom having been known for over 60 years, this is the first evidence that venom-secreted NGF follows the molecular evolutionary pattern of other venom components, and thus likely participates in prey-envenomation.

Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matte, Allan; Grosse, Stephan; Bergeron, Hélène

The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavoring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a -barrel structure and two -helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally relatedmore » proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the -barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site.« less

Influence of the Envelope gp120 Phe 43 Cavity on HIV-1 Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses

PubMed Central

Prévost, Jérémie; Zoubchenok, Daria; Richard, Jonathan; Veillette, Maxime; Pacheco, Beatriz; Coutu, Mathieu; Brassard, Nathalie; Parsons, Matthew S.; Ruxrungtham, Kiat; Bunupuradah, Torsak; Tovanabutra, Sodsai; Hwang, Kwan-Ki; Moody, M. Anthony; Haynes, Barton F.; Bonsignori, Mattia; Sodroski, Joseph; Kaufmann, Daniel E.; Shaw, George M.; Chenine, Agnès L.

2017-01-01

ABSTRACT HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy. IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV-positive (HIV+) sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43 cavity-filling His 375 residue, which increases the propensity of Env to sample the CD4-bound conformation, thereby increasing susceptibility to ADCC. PMID:28100618

Crystal structures of two tropinone reductases: Different reaction stereospecificities in the same protein fold

PubMed Central

Nakajima, Keiji; Yamashita, Atsuko; Akama, Hiroyuki; Nakatsu, Toru; Kato, Hiroaki; Hashimoto, Takashi; Oda, Jun’ichi; Yamada, Yasuyuki

1998-01-01

A pair of tropinone reductases (TRs) share 64% of the same amino acid residues and belong to the short-chain dehydrogenase/reductase family. In the synthesis of tropane alkaloids in several medicinal plants, the TRs reduce a carbonyl group of an alkaloid intermediate, tropinone, to hydroxy groups with different diastereomeric configurations. To clarify the structural basis for their different reaction stereospecificities, we determined the crystal structures of the two enzymes at 2.4- and 2.3-Å resolutions. The overall folding of the two enzymes was almost identical. The conservation was not confined within the core domains that are conserved within the protein family but extended outside the core domain where each family member has its characteristic structure. The binding sites for the cofactor and the positions of the active site residues were well conserved between the two TRs. The substrate binding site was composed mostly of hydrophobic amino acids in both TRs, but the presence of different charged residues conferred different electrostatic environments on the two enzymes. A modeling study indicated that these charged residues play a major role in controlling the binding orientation of tropinone within the substrate binding site, thereby determining the stereospecificity of the reaction product. The results obtained herein raise the possibility that in certain cases different stereospecificities can be acquired in enzymes by changing a few amino acid residues within substrate binding sites. PMID:9560196

Crystal Structure of Botulinum Neurotoxin Type a in Complex With the Cell Surface Co-Receptor GT1b-Insight Into the Toxin-Neuron Interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stenmark, P.; Dupuy, J.; Inamura, A.

2009-05-26

Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in themore » toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.« less

Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

PubMed Central

Garcin, Elsa D.; Arvai, Andrew S.; Rosenfeld, Robin J.; Kroeger, Matt D.; Crane, Brian R.; Andersson, Gunilla; Andrews, Glen; Hamley, Peter J.; Mallinder, Philip R.; Nicholls, David J.; St-Gallay, Stephen A.; Tinker, Alan C.; Gensmantel, Nigel P.; Mete, Antonio; Cheshire, David R.; Connolly, Stephen; Stuehr, Dennis J.; Åberg, Anders; Wallace, Alan V.; Tainer, John A.; Getzoff, Elizabeth D.

2008-01-01

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low levels, and a defensive cytotoxin at higher levels. The high active-site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock, and cancer. Our structural and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a novel specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents towards remote specificity pockets, accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active-site conservation. PMID:18849972

Characterization of a novel domain ‘GATE’ in the ABC protein DrrA and its role in drug efflux by the DrrAB complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Han; Rahman, Sadia; Li, Wen

2015-03-27

A novel domain, GATE (Glycine-loop And Transducer Element), is identified in the ABC protein DrrA. This domain shows sequence and structural conservation among close homologs of DrrA as well as distantly-related ABC proteins. Among the highly conserved residues in this domain are three glycines, G215, G221 and G231, of which G215 was found to be critical for stable expression of the DrrAB complex. Other conserved residues, including E201, G221, K227 and G231, were found to be critical for the catalytic and transport functions of the DrrAB transporter. Structural analysis of both the previously published crystal structure of the DrrA homologmore » MalK and the modeled structure of DrrA showed that G215 makes close contacts with residues in and around the Walker A motif, suggesting that these interactions may be critical for maintaining the integrity of the ATP binding pocket as well as the complex. It is also shown that G215A or K227R mutation diminishes some of the atomic interactions essential for ATP catalysis and overall transport function. Therefore, based on both the biochemical and structural analyses, it is proposed that the GATE domain, located outside of the previously identified ATP binding and hydrolysis motifs, is an additional element involved in ATP catalysis. - Highlights: • A novel domain ‘GATE’ is identified in the ABC protein DrrA. • GATE shows high sequence and structural conservation among diverse ABC proteins. • GATE is located outside of the previously studied ATP binding and hydrolysis motifs. • Conserved GATE residues are critical for stability of DrrAB and for ATP catalysis.« less

Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

DOE PAGES

Smith, Steven D.; Bridou, Romain; Johs, Alexander; ...

2015-02-27

Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17

DOE Office of Scientific and Technical Information (OSTI.GOV)

Czerny, Daniel D.; Padmanaban, Senthilkumar; Anishkin, Andriy

Using Arabidopsis thaliana AtCHX17 as an example, we combine structural modeling and mutagenesis to provide insights on its protein architecture and transport function which is poorly characterized. This approach is based on the observation that protein structures are significantly more conserved in evolution than linear sequences, and mechanistic similarities among diverse transporters are emerging. Two homology models of AtCHX17 were obtained that show a protein fold similar to known structures of bacterial Na +/H + antiporters, EcNhaA and TtNapA. The distinct secondary and tertiary structure models highlighted residues at positions potentially important for CHX17 activity. Mutagenesis showed that asparagine-N200 andmore » aspartate-D201 inside transmembrane5 (TM5), and lysine-K355 inside TM10 are critical for AtCHX17 activity. We reveal previously unrecognized threonine-T170 and lysine-K383 as key residues at unwound regions in the middle of TM4 and TM11 α-helices, respectively. Mutation of glutamate-E111 located near the membrane surface inhibited AtCHX17 activity, suggesting a role in pH sensing. The long carboxylic tail of unknown purpose has an alternating β-sheet and α-helix secondary structure that is conserved in prokaryote universal stress proteins. Here, these results support the overall architecture of AtCHX17 and identify D201, N200 and novel residues T170 and K383 at the functional core which likely participates in ion recognition, coordination and/or translocation, similar to characterized cation/H + exchangers. The core of AtCHX17 models according to EcNhaA and TtNapA templates faces inward and outward, respectively, which may reflect two conformational states of the alternating access transport mode for proteins belonging to the plant CHX family.« less

Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17

DOE PAGES

Czerny, Daniel D.; Padmanaban, Senthilkumar; Anishkin, Andriy; ...

2016-05-11

Using Arabidopsis thaliana AtCHX17 as an example, we combine structural modeling and mutagenesis to provide insights on its protein architecture and transport function which is poorly characterized. This approach is based on the observation that protein structures are significantly more conserved in evolution than linear sequences, and mechanistic similarities among diverse transporters are emerging. Two homology models of AtCHX17 were obtained that show a protein fold similar to known structures of bacterial Na +/H + antiporters, EcNhaA and TtNapA. The distinct secondary and tertiary structure models highlighted residues at positions potentially important for CHX17 activity. Mutagenesis showed that asparagine-N200 andmore » aspartate-D201 inside transmembrane5 (TM5), and lysine-K355 inside TM10 are critical for AtCHX17 activity. We reveal previously unrecognized threonine-T170 and lysine-K383 as key residues at unwound regions in the middle of TM4 and TM11 α-helices, respectively. Mutation of glutamate-E111 located near the membrane surface inhibited AtCHX17 activity, suggesting a role in pH sensing. The long carboxylic tail of unknown purpose has an alternating β-sheet and α-helix secondary structure that is conserved in prokaryote universal stress proteins. Here, these results support the overall architecture of AtCHX17 and identify D201, N200 and novel residues T170 and K383 at the functional core which likely participates in ion recognition, coordination and/or translocation, similar to characterized cation/H + exchangers. The core of AtCHX17 models according to EcNhaA and TtNapA templates faces inward and outward, respectively, which may reflect two conformational states of the alternating access transport mode for proteins belonging to the plant CHX family.« less

Specific Function of the Met-Tyr-Trp Adduct Radical and Residues Arg-418 and Asp-137 in the Atypical Catalase Reaction of Catalase-Peroxidase KatG*

PubMed Central

Zhao, Xiangbo; Khajo, Abdelahad; Jarrett, Sanchez; Suarez, Javier; Levitsky, Yan; Burger, Richard M.; Jarzecki, Andrzej A.; Magliozzo, Richard S.

2012-01-01

Catalase activity of the dual-function heme enzyme catalase-peroxidase (KatG) depends on several structural elements, including a unique adduct formed from covalently linked side chains of three conserved amino acids (Met-255, Tyr-229, and Trp-107, Mycobacterium tuberculosis KatG numbering) (MYW). Mutagenesis, electron paramagnetic resonance, and optical stopped-flow experiments, along with calculations using density functional theory (DFT) methods revealed the basis of the requirement for a radical on the MYW-adduct, for oxyferrous heme, and for conserved residues Arg-418 and Asp-137 in the rapid catalase reaction. The participation of an oxyferrous heme intermediate (dioxyheme) throughout the pH range of catalase activity is suggested from our finding that carbon monoxide inhibits the activity at both acidic and alkaline pH. In the presence of H2O2, the MYW-adduct radical is formed normally in KatG[D137S] but this mutant is defective in forming dioxyheme and lacks catalase activity. KatG[R418L] is also catalase deficient but exhibits normal formation of the adduct radical and dioxyheme. Both mutants exhibit a coincidence between MYW-adduct radical persistence and H2O2 consumption as a function of time, and enhanced subunit oligomerization during turnover, suggesting that the two mutations disrupting catalase turnover allow increased migration of the MYW-adduct radical to protein surface residues. DFT calculations showed that an interaction between the side chain of residue Arg-418 and Tyr-229 in the MYW-adduct radical favors reaction of the radical with the adjacent dioxyheme intermediate present throughout turnover in WT KatG. Release of molecular oxygen and regeneration of resting enzyme are thereby catalyzed in the last step of a proposed catalase reaction. PMID:22918833

An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor.

PubMed

Urizar, Eneko; Claeysen, Sylvie; Deupí, Xavier; Govaerts, Cedric; Costagliola, Sabine; Vassart, Gilbert; Pardo, Leonardo

2005-04-29

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.

Subsurface Manure Application for Conservation Tillage and Pasture Soils and Their Impact on the Nitrogen Balance

USDA-ARS?s Scientific Manuscript database

Incorporating manures into soil with conventional tillage is an effective means to reduce ammonia volatilization and conserve manure nitrogen. However, it is not possible in pasture and is not readily compatible with high-residue soil conservation practices for rowcrops. A variety of manure injecto...

Conservation of hot regions in protein-protein interaction in evolution.

PubMed

Hu, Jing; Li, Jiarui; Chen, Nansheng; Zhang, Xiaolong

2016-11-01

The hot regions of protein-protein interactions refer to the active area which formed by those most important residues to protein combination process. With the research development on protein interactions, lots of predicted hot regions can be discovered efficiently by intelligent computing methods, while performing biology experiments to verify each every prediction is hardly to be done due to the time-cost and the complexity of the experiment. This study based on the research of hot spot residue conservations, the proposed method is used to verify authenticity of predicted hot regions that using machine learning algorithm combined with protein's biological features and sequence conservation, though multiple sequence alignment, module substitute matrix and sequence similarity to create conservation scoring algorithm, and then using threshold module to verify the conservation tendency of hot regions in evolution. This research work gives an effective method to verify predicted hot regions in protein-protein interactions, which also provides a useful way to deeply investigate the functional activities of protein hot regions. Copyright © 2016. Published by Elsevier Inc.

Influence of hydrophobic and superhydrophobic surfaces on reducing aerodynamic insect residues

NASA Astrophysics Data System (ADS)

Krishnan, K. Ghokulla; Milionis, Athanasios; Loth, Eric; Farrell, Thomas E.; Crouch, Jeffrey D.; Berry, Douglas H.

2017-01-01

Insect fouling during takeoff, climb and landing can result in increased drag and fuel consumption for aircrafts with laminar-flow surfaces. This study investigates the effectiveness of various hydrophobic and superhydrophobic surfaces in reducing residue of insects on an aerodynamic surface at relatively high impact speeds (about 45 m/s). An experimental setup consisting of a wind tunnel and a method to inject live flightless fruit flies was used to test the effectiveness of various surfaces against insect fouling. Insect fouling was analyzed based on residue area and height from multiple impacts. In general most of the residue area was due to the hemolymph spreading while most of the residue height was due to adhesion of exoskeleton parts. Hydrophobic and especially superhydrophobic surfaces performed better than a hydrophilic aluminum surface in terms of minimizing the residue area of various insect components (exoskeleton, hemolymph, and red fluid). Surfaces with reduced wettability and short lateral length scales tended to have the smallest residue area. Residue height was not as strongly influenced by surface wettability since even a single exoskeleton adhered to the surface upon impact was enough to produce a residue height of the order of one mm. In general, the results indicate that hemolymph spread needs to be avoided (e.g. by having reduced wettability and short lateral correlation lengths) in order to minimize the residue area, while exoskeleton adherence needs to be avoided (e.g. by having oleophobic properties and micro/nano roughness) in order to minimize the residue height. In particular, two of the superhydrophobic coatings produced substantial reduction in residue height and area, relative to the baseline surface of aluminum. However, the surfaces also showed poor mechanical durability on the high-speed insect impact location. This suggests that although low wettability materials show great insect anti-fouling behavior, their durability needs to be substantially improved in order to withstand harsh aerospace conditions.

Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Sun; Li, Shu-Xing; Bren, Nina

2013-09-01

To identify high-affinity interactions between long-chain α-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between α-btx (α-bungarotoxin) and a pentameric ligand-binding domain constructed from the human α7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 Å 2 (1 Å=0.1 nm) of surface area, within which Arg 36 and Phe 32 from finger II of α-btx form a π-cation stack that aligns edge-to-face with the conserved Tyr 184 from loop-C of α7, while Asp 30 of α-btx forms a hydrogen bond with the hydroxy group of Tyr 184. These inter-residue interactions diverge from thosemore » in a 4.2 Å structure of α-ctx (α-cobratoxin) bound to AChBP, but are similar to those in a 1.94 Å structure of α-btx bound to the monomeric α1 extracellular domain, although compared with the monomer-bound complex, the α-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr 184 with a threonine residue abolishes high-affinity α-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the α-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings highlight structural principles by which α-neurotoxins interact with nicotinic receptors.« less

75 FR 35127 - Hazardous and Solid Waste Management System; Identification and Listing of Special Wastes...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-21

...The Environmental Protection Agency (EPA or Agency) is proposing to regulate for the first time, coal combustion residuals (CCRs) under the Resource Conservation and Recovery Act (RCRA) to address the risks from the disposal of CCRs generated from the combustion of coal at electric utilities and independent power producers. However, the Agency is considering two options in this proposal and, thus, is proposing two alternative regulations. Under the first proposal, EPA would reverse its August 1993 and May 2000 Bevill Regulatory Determinations regarding coal combustion residuals (CCRs) and list these residuals as special wastes subject to regulation under subtitle C of RCRA, when they are destined for disposal in landfills or surface impoundments. Under the second proposal, EPA would leave the Bevill determination in place and regulate disposal of such materials under subtitle D of RCRA by issuing national minimum criteria. Under both alternatives EPA is proposing to establish dam safety requirements to address the structural integrity of surface impoundments to prevent catastrophic releases. EPA is not proposing to change the May 2000 Regulatory Determination for beneficially used CCRs, which are currently exempt from the hazardous waste regulations under Section 3001(b)(3)(A) of RCRA. However, EPA is clarifying this determination and seeking comment on potential refinements for certain beneficial uses. EPA is also not proposing to address the placement of CCRs in mines, or non-minefill uses of CCRs at coal mine sites in this action.

A feature-based approach to modeling protein-protein interaction hot spots.

PubMed

Cho, Kyu-il; Kim, Dongsup; Lee, Doheon

2009-05-01

Identifying features that effectively represent the energetic contribution of an individual interface residue to the interactions between proteins remains problematic. Here, we present several new features and show that they are more effective than conventional features. By combining the proposed features with conventional features, we develop a predictive model for interaction hot spots. Initially, 54 multifaceted features, composed of different levels of information including structure, sequence and molecular interaction information, are quantified. Then, to identify the best subset of features for predicting hot spots, feature selection is performed using a decision tree. Based on the selected features, a predictive model for hot spots is created using support vector machine (SVM) and tested on an independent test set. Our model shows better overall predictive accuracy than previous methods such as the alanine scanning methods Robetta and FOLDEF, and the knowledge-based method KFC. Subsequent analysis yields several findings about hot spots. As expected, hot spots have a larger relative surface area burial and are more hydrophobic than other residues. Unexpectedly, however, residue conservation displays a rather complicated tendency depending on the types of protein complexes, indicating that this feature is not good for identifying hot spots. Of the selected features, the weighted atomic packing density, relative surface area burial and weighted hydrophobicity are the top 3, with the weighted atomic packing density proving to be the most effective feature for predicting hot spots. Notably, we find that hot spots are closely related to pi-related interactions, especially pi . . . pi interactions.

Progress on New Hepatitis C Virus Targets: NS2 and NS5A

NASA Astrophysics Data System (ADS)

Marcotrigiano, Joseph

Hepatitis C virus (HCV) is a major global health problem, affecting about 170 million people worldwide. Chronic infection can lead to cirrhosis and liver cancer. The replication machine of HCV is a multi-subunit membrane associated complex, consisting of nonstructural proteins (NS2-5B), which replicate the viral RNA genome. The structures of NS5A and NS2 were recently determined. NS5A is an essential replicase component that also modulates numerous cellular processes ranging from innate immunity to cell growth and survival. The structure reveals a novel protein fold, a new zinc coordination motif, a disulfide bond and a dimer interface. Analysis of molecular surfaces suggests the location of the membrane interaction surface of NS5A, as well as hypothetical protein and RNA binding sites. NS2 is one of two virally encoded proteases that are required for processing the viral polyprotein into the mature nonstructural proteins. NS2 is a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer and the nucleophilic cysteine by the other. The C-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. The structure also reveals possible sites of membrane interaction, a rare cis-proline residue, and highly conserved dimer contacts. The novel features of both structures have changed the current view of HCV polyprotein replication and present new opportunities for antiviral drug design.

The X-ray Crystallographic Structure and Activity Analysis of a Pseudomonas-Specific Subfamily of the HAD Enzyme Superfamily Evidences a Novel Biochemical Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peisach,E.; Wang, L.; Burroughs, A.

2008-01-01

The haloacid dehalogenase (HAD) superfamily is a large family of proteins dominated by phosphotransferases. Thirty-three sequence families within the HAD superfamily (HADSF) have been identified to assist in function assignment. One such family includes the enzyme phosphoacetaldehyde hydrolase (phosphonatase). Phosphonatase possesses the conserved Rossmanniod core domain and a C1-type cap domain. Other members of this family do not possess a cap domain and because the cap domain of phosphonatase plays an important role in active site desolvation and catalysis, the function of the capless family members must be unique. A representative of the capless subfamily, PSPTO{_}2114, from the plant pathogenmore » Pseudomonas syringae, was targeted for catalytic activity and structure analyses. The X-ray structure of PSPTO{_}2114 reveals a capless homodimer that conserves some but not all of the intersubunit contacts contributed by the core domains of the phosphonatase homodimer. The region of the PSPTO{_}2114 that corresponds to the catalytic scaffold of phosphonatase (and other HAD phosphotransfereases) positions amino acid residues that are ill suited for Mg+2 cofactor binding and mediation of phosphoryl group transfer between donor and acceptor substrates. The absence of phosphotransferase activity in PSPTO{_}2114 was confirmed by kinetic assays. To explore PSPTO{_}2114 function, the conservation of sequence motifs extending outside of the HADSF catalytic scaffold was examined. The stringently conserved residues among PSPTO{_}2114 homologs were mapped onto the PSPTO{_}2114 three-dimensional structure to identify a surface region unique to the family members that do not possess a cap domain. The hypothesis that this region is used in protein-protein recognition is explored to define, for the first time, HADSF proteins which have acquired a function other than that of a catalyst. Proteins 2008.« less

Analysis of the Effects of Polymorphism on Pollen Profilin Structural Functionality and the Generation of Conformational, T- and B-Cell Epitopes

PubMed Central

Jimenez-Lopez, Jose C.; Rodríguez-García, María I.; Alché, Juan D.

2013-01-01

An extensive polymorphism analysis of pollen profilin, a fundamental regulator of the actin cytoskeleton dynamics, has been performed with a major focus in 3D-folding maintenance, changes in the 2-D structural elements, surface residues involved in ligands-profilin interactions and functionality, and the generation of conformational and lineal B- and T-cell epitopes variability. Our results revealed that while the general fold is conserved among profilins, substantial structural differences were found, particularly affecting the special distribution and length of different 2-D structural elements (i.e. cysteine residues), characteristic loops and coils, and numerous micro-heterogeneities present in fundamental residues directly involved in the interacting motifs, and to some extension these residues nearby to the ligand-interacting areas. Differential changes as result of polymorphism might contribute to generate functional variability among the plethora of profilin isoforms present in the olive pollen from different genetic background (olive cultivars), and between plant species, since biochemical interacting properties and binding affinities to natural ligands may be affected, particularly the interactions with different actin isoforms and phosphoinositides lipids species. Furthermore, conspicuous variability in lineal and conformational epitopes was found between profilins belonging to the same olive cultivar, and among different cultivars as direct implication of sequences polymorphism. The variability of the residues taking part of IgE-binding epitopes might be the final responsible of the differences in cross-reactivity among olive pollen cultivars, among pollen and plant-derived food allergens, as well as between distantly related pollen species, leading to a variable range of allergy reactions among atopic patients. Identification and analysis of commonly shared and specific epitopes in profilin isoforms is essential to gain knowledge about the interacting surface of these epitopes, and for a better understanding of immune responses, helping design and development of rational and effective immunotherapy strategies for the treatment of allergy diseases. PMID:24146818

[Thoracoscopic diagnosis and treatment of postoperative residual cavities].

PubMed

Ioffe, D Ts; Dashiev, V A; Amanov, S A

1987-03-01

Investigations performed in 41 patients with postoperative residual cavities after surgical interventions of different volume have shown high value of thoracoscopy as an additional diagnostic and curative method. The endoscopy findings determinate further curative tactics--surgery or conservative therapy.

Proteins feel more than they see: fine-tuning of binding affinity by properties of the non-interacting surface.

PubMed

Kastritis, Panagiotis L; Rodrigues, João P G L M; Folkers, Gert E; Boelens, Rolf; Bonvin, Alexandre M J J

2014-07-15

Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct contributions from the interface, the non-interacting surface (NIS) also plays an important role in binding affinity, especially polar and charged residues. Their percentage on the NIS is conserved over orthologous complexes indicating an evolutionary selection pressure. Their effect on binding affinity can be explained by long-range electrostatic contributions and surface-solvent interactions that are known to determine the local frustration of the protein complex surface. Including these in a simple model significantly improves the affinity prediction of protein complexes from structural models. The impact of mutations outside the interacting surface on binding affinity is supported by experimental alanine scanning mutagenesis data. These results enable the development of more sophisticated and integrated biophysical models of binding affinity and open new directions in experimental control and modulation of biomolecular interactions. Copyright © 2014. Published by Elsevier Ltd.

Structural Basis for the De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine in Gram-positive Bacteria*

PubMed Central

Little, Dustin J.; Bamford, Natalie C.; Pokrovskaya, Varvara; Robinson, Howard; Nitz, Mark; Howell, P. Lynne

2014-01-01

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In staphylococci, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the extracellular protein IcaB is required for biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of IcaB from Ammonifex degensii (IcaBAd) has been determined to 1.7 Å resolution. The structure of IcaBAd reveals a (β/α)7 barrel common to the family four carbohydrate esterases (CE4s) with the canonical motifs circularly permuted. The metal dependence of IcaBAd is similar to most CE4s showing the maximum rates of de-N-acetylation with Ni2+, Co2+, and Zn2+. From docking studies with β-1,6-GlcNAc oligomers and structural comparison to PgaB from Escherichia coli, the Gram-negative homologue of IcaB, we identify Arg-45, Tyr-67, and Trp-180 as key residues for PNAG binding during catalysis. The absence of these residues in PgaB provides a rationale for the requirement of a C-terminal domain for efficient deacetylation of PNAG in Gram-negative species. Mutational analysis of conserved active site residues suggests that IcaB uses an altered catalytic mechanism in comparison to other characterized CE4 members. Furthermore, we identified a conserved surface-exposed hydrophobic loop found only in Gram-positive homologues of IcaB. Our data suggest that this loop is required for membrane association and likely anchors IcaB to the membrane during polysaccharide biosynthesis. The work presented herein will help guide the design of IcaB inhibitors to combat biofilm formation by staphylococci. PMID:25359777

The electrostatic role of the Zn-Cys2His2 complex in binding of operator DNA with transcription factors: mouse EGR-1 from the Cys2His2 family.

PubMed

Chirgadze, Y N; Boshkova, E A; Polozov, R V; Sivozhelezov, V S; Dzyabchenko, A V; Kuzminsky, M B; Stepanenko, V A; Ivanov, V V

2018-01-07

The mouse factor Zif268, known also as early growth response protein EGR-1, is a classical representative for the Cys2His2 transcription factor family. It is required for binding the RNA polymerase with operator dsDNA to initialize the transcription process. We have shown that only in this family of total six Zn-finger protein families the Zn complex plays a significant role in the protein-DNA binding. Electrostatic feature of this complex in the binding of factor Zif268 from Mus musculus with operator DNA has been considered. The factor consists of three similar Zn-finger units which bind with triplets of coding DNA. Essential contacts of the factor with the DNA phosphates are formed by three conservative His residues, one in each finger. We describe here the results of calculations of the electrostatic potentials for the Zn-Cys2His2 complex, Zn-finger unit 1, and the whole transcription factor. The potential of Zif268 has a positive area on the factor surface, and it corresponds exactly to the binding sites of each of Zn-finger units. The main part of these areas is determined by conservative His residues, which form contacts with the DNA phosphate groups. Our result shows that the electrostatic positive potential of this histidine residue is enhanced due to the Zn complex. The other contacts of the Zn-finger with DNA are related to nucleotide bases, and they are responsible for the sequence-specific binding with DNA. This result may be extended to all other members of the Cys2His2 transcription factor family.

Purification of an alpha amylase from Aspergillus flavus NSH9 and molecular characterization of its nucleotide gene sequence.

PubMed

Karim, Kazi Muhammad Rezaul; Husaini, Ahmad; Sing, Ngieng Ngui; Sinang, Fazia Mohd; Roslan, Hairul Azman; Hussain, Hasnain

2018-04-01

In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca 2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca 2+ -binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.

Evolutionary conservativeness of electric field in the Cu,Zn superoxide dismutase active site. Evidence for co-ordinated mutation of charged amino acid residues.

PubMed

Desideri, A; Falconi, M; Polticelli, F; Bolognesi, M; Djinovic, K; Rotilio, G

1992-01-05

Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast, and the isoenzymes A and B from the amphibian Xenopus laevis. The three-dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template. The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X. laevis isoenzymes. The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases. These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities. This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family.

Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Backovic, Marija; Longnecker, Richard; Jardetzky, Theodore S

Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which formsmore » 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.« less

Diversity of environmental single-stranded DNA phages revealed by PCR amplification of the partial major capsid protein

PubMed Central

Hopkins, Max; Kailasan, Shweta; Cohen, Allison; Roux, Simon; Tucker, Kimberly Pause; Shevenell, Amelia; Agbandje-McKenna, Mavis; Breitbart, Mya

2014-01-01

The small single-stranded DNA (ssDNA) bacteriophages of the subfamily Gokushovirinae were traditionally perceived as narrowly targeted, niche-specific viruses infecting obligate parasitic bacteria, such as Chlamydia. The advent of metagenomics revealed gokushoviruses to be widespread in global environmental samples. This study expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from 10 environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. Residue-level conservation data generated from multiple alignments was combined with a predicted 3D structure, revealing a tendency for structurally internal residues to be more highly conserved than surface-presenting protein–protein or viral–host interaction domains. Aggregating this data set into a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different samples. Antarctic sediment samples contained the most diverse gokushovirus communities, whereas freshwater springs from Florida were the least diverse. Whether the observed diversity is being driven by environmental factors or host-binding interactions remains an open question. The high environmental diversity of this previously overlooked ssDNA viral group necessitates further research elucidating their natural hosts and exploring their ecological roles. PMID:24694711

[Family of ribosomal proteins S1 contains unique conservative domain].

PubMed

Deriusheva, E I; Machulin, A V; Selivanova, O M; Serdiuk, I N

2010-01-01

Different representatives of bacteria have different number of amino acid residues in the ribosomal proteins S1. This number varies from 111 (Spiroplasma kunkelii) to 863 a.a. (Treponema pallidum). Traditionally and for lack of this protein three-dimensional structure, its architecture is represented as repeating S1 domains. Number of these domains depends on the protein's length. Domain's quantity and its boundaries data are contained in the specialized databases, such as SMART, Pfam and PROSITE. However, for the same object these data may be very different. For search of domain's quantity and its boundaries, new approach, based on the analysis of dicted secondary structure (PsiPred), was used. This approach allowed us to reveal structural domains in amino acid sequences of S1 proteins and at that number varied from one to six. Alignment of S1 proteins, containing different domain's number, with the S1 RNAbinding domain of Escherichia coli PNPase elicited a fact that in family of ribosomal proteins SI one domain has maximal homology with S1 domain from PNPase. This conservative domain migrates along polypeptide chain and locates in proteins, containing different domain's number, according to specified pattern. In this domain as well in the S1 domain from PNPase, residues Phe-19, Phe-22, His-34, Asp-64 and Arg-68 are clustered on the surface and formed RNA binding site.

Characterization of the Role of a Highly Conserved Sequence in ATP Binding Cassette Transporter G (ABCG) Family in ABCG1 Stability, Oligomerization, and Trafficking

PubMed Central

2013-01-01

ATP-binding cassette transporter G1 (ABCG1) mediates cholesterol and oxysterol efflux onto lipidated lipoproteins and plays an important role in macrophage reverse cholesterol transport. Here, we identified a highly conserved sequence present in the five ABCG transporter family members. The conserved sequence is located between the nucleotide binding domain and the transmembrane domain and contains five amino acid residues from Asn at position 316 to Phe at position 320 in ABCG1 (NPADF). We found that cells expressing mutant ABCG1, in which Asn316, Pro317, Asp319, and Phe320 in the conserved sequence were replaced with Ala simultaneously, showed impaired cholesterol efflux activity compared with wild type ABCG1-expressing cells. A more detailed mutagenesis study revealed that mutation of Asn316 or Phe 320 to Ala significantly reduced cellular cholesterol and 7-ketocholesterol efflux conferred by ABCG1, whereas replacement of Pro317 or Asp319 with Ala had no detectable effect. To confirm the important role of Asn316 and Phe320, we mutated Asn316 to Asp (N316D) and Gln (N316Q), and Phe320 to Ile (F320I) and Tyr (F320Y). The mutant F320Y showed the same phenotype as wild type ABCG1. However, the efflux of cholesterol and 7-ketocholesterol was reduced in cells expressing ABCG1 mutant N316D, N316Q, or F320I compared with wild type ABCG1. Further, mutations N316Q and F320I impaired ABCG1 trafficking while having no marked effect on the stability and oligomerization of ABCG1. The mutant N316Q and F320I could not be transported to the cell surface efficiently. Instead, the mutant proteins were mainly localized intracellularly. Thus, these findings indicate that the two highly conserved amino acid residues, Asn and Phe, play an important role in ABCG1-dependent export of cellular cholesterol, mainly through the regulation of ABCG1 trafficking. PMID:24320932

Aromaticity at the water-hydrocarbon core interface of the membrane

PubMed Central

Lizardi-Ortiz, José E.; Hyzinski-García, María C.; Fernández-Gerena, José L.; Osorio-Martínez, Karen M.; Velázquez-Rivera, Eric; Valle-Avilés, Félix L.; Lasalde-Dominicci, José A.

2011-01-01

Almost all lipid-exposed transmembrane domains of integral proteins contain aromatic residues flanking the hydrophobic segment of the domains. These residues generally reside close to the carbonyl region of the membrane, and several structural and functional roles have been associated to these residues. Although the roles and physicochemical reasons for aromatic preference have been extensively studied using model systems, few studies have been done in a native membrane system. To gain insight about the mechanistic implication for this aromatic preference, we selected position αF426 of the muscle-type nicotinic acetylcholine receptor (nAChR). αF426 is a lipid-exposed residue at the extracellular segment of the αM4 transmembrane domain and is highly conserved among different nAChR subunits and species. We used site-directed mutagenesis, α-Bungarotoxin-binding assay, and two-electrodes voltage clamp in Xenopus laevis oocytes to characterize mutations at position αF426, which impart different physicochemical properties like volume, polarity, hydrogen bonds, aromaticity and net electrical charge. All mutations except the aromatic residues resulted in a significant reduction of the nAChR cell-surface levels and the macroscopic currents to acetylcholine. These results suggest that position αF426 contributes to structural stability and open-close transitions of the nAChR. Finally, the present study also provides information about how intermolecular interactions at position α426 modulate open-close transitions of the nAChR. PMID:18836298

Crystal structure and biochemical features of dye-decolorizing peroxidase YfeX from Escherichia coli O157 Asp{sup 143} and Arg{sup 232} play divergent roles toward different substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xiuhua; Yuan, Zenglin; Wang, Jiaxu

YfeX from Escherichia coli O157 is a bacterial dye-decolorizing peroxidase that represents both dye-decoloring activity and typical peroxidase activity. We reported the crystal structure of YfeX bound to heme at 2.09 Å resolution. The YfeX monomer resembles a ferredoxin-like fold and contains two domains. The three conserved residues surrounding the heme group are His{sup 215}, Asp{sup 143} and Arg{sup 232}. His{sup 215} functions as the proximal axial ligand of the heme iron atom. Biochemical data show that the catalytic significance of the conserved Asp{sup 143} and Arg{sup 232} depends on the substrate types and that YfeX may adopt various catalytic mechanismsmore » toward divergent substrates. In addition, it is observed that an access tunnel spans from the protein molecular surface to the heme distal region, it serves as the passageway for the entrance and binding of the H{sub 2}O{sub 2}. - Highlights: • We determined the crystal structure of YfeX bound to heme at 2.09 Å resolution. • The conserved Asp{sup 143} and Arg{sup 232} play different roles toward divergent substrates. • The H{sub 2}O{sub 2} access tunnel spans from the protein molecular surface to the heme distal region.« less

Interactions between the Nse3 and Nse4 Components of the SMC5-6 Complex Identify Evolutionarily Conserved Interactions between MAGE and EID Families

PubMed Central

Kozakova, Lucie; Liao, Chunyan; Guerineau, Marc; Colnaghi, Rita; Vidot, Susanne; Marek, Jaromir; Bathula, Sreenivas R.; Lehmann, Alan R.; Palecek, Jan

2011-01-01

Background The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6–8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors. Methodology/Principal Findings Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins. Conclusions/Significance We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins. PMID:21364888

Herpes Simplex Virus 1 UL37 Protein Tyrosine Residues Conserved among All Alphaherpesviruses Are Required for Interactions with Glycoprotein K, Cytoplasmic Virion Envelopment, and Infectious Virus Production

PubMed Central

Chouljenko, Dmitry V.; Jambunathan, Nithya; Chouljenko, Vladimir N.; Naderi, Misagh; Brylinski, Michal; Caskey, John R.

2016-01-01

ABSTRACT The herpes simplex virus 1 (HSV-1) UL37 protein functions in virion envelopment at trans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K (gK) and its interacting partner protein UL20 (N. Jambunathan, D. Chouljenko, P. Desai, A. S. Charles, R. Subramanian, V. N. Chouljenko, and K. G. Kousoulas, J Virol 88:5927–5935, 2014, http://dx.doi.org/10.1128/JVI.00278-14), facilitating cytoplasmic virion envelopment. Alignment of UL37 homologs encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented the virus efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477, and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines with alanines substantially reduced the ability of gK to interact with UL37. Alignment of HSV UL37 with the human cytomegalovirus and Epstein-Barr virus UL37 homologs revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that the UL37 conserved tyrosine 480 residue plays a crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production. IMPORTANCE The HSV-1 UL37 protein is conserved among all herpesviruses, functions in both retrograde and anterograde transport of virion capsids, and plays critical roles in cytoplasmic virion envelopment by interacting with gK. We show here that UL37 tyrosine residues conserved among all alphaherpesviruses serve critical roles in cytoplasmic virion envelopment and interactions with gK. PMID:27630233

Evolutionary analysis of FAM83H in vertebrates.

PubMed

Huang, Wushuang; Yang, Mei; Wang, Changning; Song, Yaling

2017-01-01

Amelogenesis imperfecta is a group of disorders causing abnormalities in enamel formation in various phenotypes. Many mutations in the FAM83H gene have been identified to result in autosomal dominant hypocalcified amelogenesis imperfecta in different populations. However, the structure and function of FAM83H and its pathological mechanism have yet to be further explored. Evolutionary analysis is an alternative for revealing residues or motifs that are important for protein function. In the present study, we chose 50 vertebrate species in public databases representative of approximately 230 million years of evolution, including 1 amphibian, 2 fishes, 7 sauropsidas and 40 mammals, and we performed evolutionary analysis on the FAM83H protein. By sequence alignment, conserved residues and motifs were indicated, and the loss of important residues and motifs of five special species (Malayan pangolin, platypus, minke whale, nine-banded armadillo and aardvark) was discovered. A phylogenetic time tree showed the FAM83H divergent process. Positive selection sites in the C-terminus suggested that the C-terminus of FAM83H played certain adaptive roles during evolution. The results confirmed some important motifs reported in previous findings and identified some new highly conserved residues and motifs that need further investigation. The results suggest that the C-terminus of FAM83H contain key conserved regions critical to enamel formation and calcification.

The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation

PubMed Central

Didierlaurent, Ludovic; Houzet, Laurent; Morichaud, Zakia; Darlix, Jean-Luc; Mougel, Marylène

2008-01-01

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs. PMID:18641038

Influence of Surface Properties and Impact Conditions on Adhesion of Insect Residues

NASA Technical Reports Server (NTRS)

Wohl, Christopher J.; Smith, Joseph G.; Connell, John W.; Siochi, Emilie J.; Doss, Jereme R.; Shanahan, Michelle H.; Penner, Ronald K.

2015-01-01

Insect residues can cause premature transition to turbulent flow on laminar flow airfoils. Engineered surfaces that mitigate the adhesion of insect residues provide, therefore, a route to more efficient aerodynamics and reduced fuel burn rates. Areal coverage and heights of residues depend not only on surface properties, but also on impact conditions. We report high speed photography of fruit fly impacts at different angles of inclination on a rigid aluminum surface, optical microscopy and profilometry, and contact angle goniometry to support the design of engineered surfaces. For the polyurethane and epoxy coatings studied, some of which exhibited superhydrophobicity, it was determined that impact angle and surface compositions play critical roles in the efficacy of these surfaces to reduce insect residue adhesion.

Structural studies of viperin, an antiviral radical SAM enzyme.

PubMed

Fenwick, Michael K; Li, Yue; Cresswell, Peter; Modis, Yorgo; Ealick, Steven E

2017-06-27

Viperin is an IFN-inducible radical S -adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S -adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (βα) 6 -barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first β-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.

The H(2) sensor of Ralstonia eutropha: biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site.

PubMed

Buhrke, Thorsten; Brecht, Marc; Lubitz, Wolfgang; Friedrich, Bärbel

2002-09-01

[NiFe] hydrogenases contain a highly conserved histidine residue close to the [NiFe] active site which is altered by a glutamine residue in the H(2)-sensing [NiFe] hydrogenases. In this study, we exchanged the respective glutamine residue of the H(2) sensor (RH) of Ralstonia eutropha, Q67 of the RH large subunit HoxC, by histidine, asparagine and glutamate. The replacement by histidine and asparagine resulted in slightly unstable RH proteins which were hardly affected in their regulatory and enzymatic properties. The exchange to glutamate led to a completely unstable RH protein. The purified wild-type RH and the mutant protein with the Gln/His exchange were analysed by continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques. We observed a coupling of a nitrogen nucleus with the [NiFe] active site for the mutant protein which was absent in the spectrum of the wild-type RH. A combination of theoretical calculations with the experimental data provided an explanation for the observed coupling. It is shown that the coupling is due to the formation of a weak hydrogen bond between the protonated N(epsilon) nucleus of the histidine with the sulfur of a conserved cysteine residue which coordinates the metal atoms of the [NiFe] active site as a bridging ligand. The effect of this hydrogen bond on the local structure of the [NiFe] active site is discussed.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao Maofu; Kielian, Margaret

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 residuesmore » 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.« less

Assessment of interplay between UV wavelengths, material surfaces and food residues in open surface hygiene validation.

PubMed

Abban, Stephen; Jakobsen, Mogens; Jespersen, Lene

2014-12-01

The use of UV-visible radiation for detecting invisible residue on different surfaces as a means of validating cleanliness was investigated. Wavelengths at 365, 395, 435, 445, 470 and 490 nm from a monochromator were used to detect residues of beef, chicken, apple, mango and skim milk. These were on three surfaces: aluminium, fibre re-enforced plastic (FRP; Q-Liner®) and stainless steel, pre- and post a cleaning step using commercial detergent. The area covered by residues as detected by specific wavelengths was compared statistically. The sensitivity of the wavelengths for detection differed significantly (p < 0.05) for various residues depending on the material surfaces. Generally, wavelengths 365-445 nm were consistently able to illuminate all residue before cleaning, though sensitivity varied, while 490 nm showed more of the surface structural features instead of residue. The 365-395 nm wavelengths were significantly more sensitive (p < 0.05) for detecting beef and chicken residues on aluminium and stainless steel both before and after cleaning. The 435-445 nm wavelengths were significantly more sensitive for detecting apple and mango residues on the FRP both before and after cleaning. It is important when UV-systems are used as real-time tools for assessing cleanliness of surfaces that the surface materials being illuminated are taken into account in the choice of lamp wavelength, in addition to expected residue. This will ensure higher confidence in results during the use of UV-light for real-time hygiene validation of surfaces.

Surface conservation laws at microscopically diffuse interfaces.

PubMed

Chu, Kevin T; Bazant, Martin Z

2007-11-01

In studies of interfaces with dynamic chemical composition, bulk and interfacial quantities are often coupled via surface conservation laws of excess surface quantities. While this approach is easily justified for microscopically sharp interfaces, its applicability in the context of microscopically diffuse interfaces is less theoretically well-established. Furthermore, surface conservation laws (and interfacial models in general) are often derived phenomenologically rather than systematically. In this article, we first provide a mathematically rigorous justification for surface conservation laws at diffuse interfaces based on an asymptotic analysis of transport processes in the boundary layer and derive general formulae for the surface and normal fluxes that appear in surface conservation laws. Next, we use nonequilibrium thermodynamics to formulate surface conservation laws in terms of chemical potentials and provide a method for systematically deriving the structure of the interfacial layer. Finally, we derive surface conservation laws for a few examples from diffusive and electrochemical transport.

75 FR 64974 - Notice of Data Availability on Coal Combustion Residual Surface Impoundments

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-21

...-2009-0640; FRL-9216-3] RIN 2050-AE81 Notice of Data Availability on Coal Combustion Residual Surface... rulemaking (75 FR 51434, August 20, 2010) on the Disposal of Coal Combustion Residuals from Electric... Requests that EPA sent to electric utilities on their coal combustion residual surface impoundments as well...

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

USDA-ARS?s Scientific Manuscript database

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 ...

Catalytic site of human protein-glucosylgalactosylhydroxylysine glucosidase: Three crucial carboxyl residues were determined by cloning and site-directed mutagenesis.

PubMed

Hamazaki, Hideaki; Hamazaki, Michiko Horikawa

2016-01-15

Protein-glucosylgalactosylhydroxylysine glucosidase (PGGHG; EC3.2.1.107) cleaves glucose from disaccharide unit (Glc-α1,2-Gal) linked to hydroxylysine residues of collagen. In the present paper we first show that PGGHG is the product of ATHL1 gene as follows. (1) PGGHG was purified from chick embryos and digested with trypsin. LC-MS/MS analysis suggested the tryptic-peptides were from the ATHL1 gene product. (2) Chick embryo ATHL1 cDNA was cloned to a cloning and expression vector and two plasmid clones with different ATHL1 CDS insert were obtained. (3) Each plasmid DNA was transformed into Escherichia coli cells for expression and two isoforms of chicken PGGHG were obtained. (4) Both isoforms effectively released glucose from type IV collagen. Next, we searched for carboxyl residues crucial for catalytic activity as follows; human ATHL1 cDNA was cloned into a cloning and expression vector and 18 mutants were obtained by site-directed mutagenesis for 15 carboxyl residues conserved in ATHL1 of jawed vertebrates. The expression analysis indicated that substitutions of Asp301, Glu430 and Glu574 with sterically conservative (D301N, E430Q, E574Q) or functionally conservative (D301E, E430D, E574D) residues led to the complete elimination of enzyme activity. These findings lead us to the conclusion that PGGHG is encoded by ATHL1 and three carboxyl residues (corresponding to Asp301, Glu430 and Glu574 of human PGGHG) might be involved in the catalytic site of PGGHG. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

NASA Astrophysics Data System (ADS)

Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

2013-09-01

DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

Structural Insight into the Mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tchigvintsev, A.; Xu, X.; Singer, A.

2010-08-01

Cyclic diguanylate (or bis-(3'-5') cyclic dimeric guanosine monophosphate; c-di-GMP) is a ubiquitous second messenger that regulates diverse cellular functions, including motility, biofilm formation, cell cycle progression, and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TBD1265 EAL domain was determined in free state (1.8 {angstrom}) and in complex with c-di-GMP (2.35 {angstrom}), andmore » unveiled the role of conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.« less

Surface disturbance of cryptobiotic soil crusts: nitrogenase activity, chlorophyll content, and chlorophyll degradation

USGS Publications Warehouse

Belnap, Jayne; Harper, Kimball T.; Warren, Steven D.

1994-01-01

Cryptobiotic soil crusts are an important component of semiarid and arid ecosystems. An important role of these crusts is the contribution of fixed nitrogen to cold‐desert ecosystems. This study examines the residual effects of various intensities and combinations of different surface disturbances (raking, scalping, and tracked vehicles) on nitrogenase activity, chlorophyll content, and chlorophyll degradation in these soil crusts. Nine months after disturbance chlorophyll content of disturbed soils was not statistically different from undisturbed controls, except in the scalped treatments, indicating recovery of this characteristic is fairly quick unless surface material is removed. Differences in chlorophyll degradation among treatments were not statistically significant. However, nitrogenase activity in all treatments showed tremendous reductions, ranging from 77–97%, when compared to the control, indicating this characteristic is slow to recover. Consequently, assessment of crustal recovery from disturbance must include not only visual and biomass characteristics but other physiological measurements as well. Areas dominated by these crusts should be managed conservatively until the implications of crustal disturbance is better understood.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

PubMed

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Getting to the core of cadherin complex function in Caenorhabditis elegans.

PubMed

Hardin, Jeff

2015-01-01

The classic cadherin-catenin complex (CCC) mediates cell-cell adhesion in metazoans. Although substantial insights have been gained by studying the CCC in vertebrate tissue culture, analyzing requirements for and regulation of the CCC in vertebrates remains challenging. Caenorhabditis elegans is a powerful system for connecting the molecular details of CCC function with functional requirements in a living organism. Recent data, using an "angstroms to embryos" approach, have elucidated functions for key residues, conserved across all metazoans, that mediate cadherin/β-catenin binding. Other recent work reveals a novel, potentially ancestral, role for the C. elegans p120ctn homologue in regulating polarization of blastomeres in the early embryo via Cdc42 and the partitioning-defective (PAR)/atypical protein kinase C (aPKC) complex. Finally, recent work suggests that the CCC is trafficked to the cell surface via the clathrin adaptor protein complex 1 (AP-1) in surprising ways. These studies continue to underscore the value of C. elegans as a model system for identifying conserved molecular mechanisms involving the CCC.

IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE

EPA Science Inventory

Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In ...

PESTICIDE RESIDUE RECOVERIES FROM SURFACE WIPES

EPA Science Inventory

Human exposure is a consequence of pesticide use indoors with a primary source resulting from residue deposition on household surfaces. Accurate measurements of surface residues is essential for estimating exposure from different routes. Various procedures have been developed ...

Cloning and characterization of the novel D-aspartyl endopeptidase, paenidase, from Paenibacillus sp. B38.

PubMed

Nirasawa, Satoru; Nakahara, Kazuhiko; Takahashi, Saori

2018-02-27

Paenidase is the first microorganism-derived D-aspartyl endopeptidase that specifically recognizes an internal D-Asp residue to cleave [D-Asp]-X peptide bonds. Using peptide sequences obtained from the protein, we performed PCR with degenerate primers to amplify the paenidase I-encoding gene. Nucleotide sequencing revealed that mature paenidase I consists of 322 amino acid residues and that the protein is encoded as a pro-protein with a 197-amino-acid N-terminal extension compared to the mature protein. Paenidase I exhibits amino acid sequence similarity to several penicillin-binding proteins. In addition, paenidase I was classified into peptidase family S12 based on a MEROPS database search. Family S12 contains serine-type D-Ala-D-Ala carboxypeptidases that have three active site residues (Ser, Lys, and Tyr) in the conserved motifs Ser-Xaa-Thr-Lys and Tyr-Xaa-Asn. These motifs were conserved in the primary structure of paenidase I, and the role of these residues was confirmed by site-directed mutagenesis.

Kinetic and Structural Insights into the Mechanism of AMPylation by VopS Fic Domain*

PubMed Central

Luong, Phi; Kinch, Lisa N.; Brautigam, Chad A.; Grishin, Nick V.; Tomchick, Diana R.; Orth, Kim

2010-01-01

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylation of a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. Steady-state analysis of VopS mutants provides kinetic understanding on the functional role of each residue for AMPylation activity by the Fic domain. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. Discovery of a ternary reaction mechanism along with structural insight provides critical groundwork for future studies for the family of AMPylators that modify hydroxyl-containing residues with AMP. PMID:20410310

Influence of semisynthetic modification of the scaffold of a contact domain of HbS on polymerization: role of flexible surface topology in polymerization inhibition.

PubMed

Sonati, Srinivasulu; Bhutoria, Savita; Prabhakaran, Muthuchidambaran; Acharya, Seetharama A

2018-02-01

A new variant of HbS, HbS-Einstein with a deletion of segment α 23-26 in the B-helix, has been assembled by semisynthetic approach. B-helix of the α chain of cis αβ-dimer of HbS plays dominant role in the quinary interactions of deoxy HbS dimer. This B-helix is the primary scaffold that provides the orientation for the side chains of contact residues of this intermolecular contact domain. The design of HbS-Einstein has been undertaken to map the influence of perturbation of molecular surface topology and the flexibility of surface residues in the polymerization. The internal deletion exerts a strong inhibitory influence on Val-6 (β)-dependent polymerization, comparable to single contact site mutations and not for complete neutralization of Val-6(β)-dependent polymerization. The scaffold modification in cis-dimer is inhibitory, and is without any effect when present on the trans dimer. The flexibility changes in the surface topology in the region of scaffold modification apparently counteracts the intrinsic polymerization potential of the molecule. The inhibition is close to that of Le Lamentin mutation [His-20 (α) → Gln] wherein a mutation engineered without much change in flexibility of the contact domain. Interestingly, the chimeric HbS with swine-human chimeric α chain with multiple non-conservative mutations completely inhibits the Val-6(β)-dependent polymerization. The deformabilities of surface topology of chimeric HbS are comparable to HbS in spite of the multiple contact site mutations in the α-chain. We conclude that the design of antisickling Hbs for gene therapy of sickle cell disease should involve multiple mutations of intermolecular contact sites.

Conformational and functional similarities between glutaredoxin and thioredoxins.

PubMed Central

Eklund, H; Cambillau, C; Sjöberg, B M; Holmgren, A; Jörnvall, H; Höög, J O; Brändén, C I

1984-01-01

The tertiary structures of thioredoxin from Escherichia coli and bacteriophage T4 have been compared and aligned giving a common fold of 68 C alpha atoms with a root mean square difference of 2.6 A. The amino acid sequence of glutaredoxin has been aligned to those of the thioredoxins assuming that glutaredoxin has the same common fold. A model of the glutaredoxin molecule was built on a vector display using this alignment and the T4 thioredoxin tertiary structure. By comparison of the model with those of the thioredoxins, we have identified a molecular surface area on one side of the redox-active S-S bridge which we suggest is the binding area of these molecules for redox interactions with other proteins. This area comprises residues 33-34, 75-76 and 91-93 in E. coli thioredoxin; 15-16, 65-66 and 76-78 in T4 thioredoxin and 12-13, 59-60 and 69-71 in glutaredoxin. In all three molecules, this part of the surface is flat and hydrophobic. Charged groups are completely absent. In contrast, there is a cluster of charged groups on the other side of the S-S bridge which we suggest participates in the mechanisms of the redox reactions. In particular, a lysine residue close to an aromatic ring is conserved in all molecules. PMID:6378624

New insight into the architecture of oxy-anion pocket in unliganded conformation of GAT domains: A MD-simulation study.

PubMed

Bairagya, Hridoy R; Bansal, Manju

2016-03-01

Human Guanine Monophosphate Synthetase (hGMPS) converts XMP to GMP, and acts as a bifunctional enzyme with N-terminal "glutaminase" (GAT) and C-terminal "synthetase" domain. The enzyme is identified as a potential target for anti-cancer and immunosuppressive therapies. GAT domain of enzyme plays central role in metabolism, and contains conserved catalytic residues Cys104, His190, and Glu192. MD simulation studies on GAT domain suggest that position of oxyanion in unliganded conformation is occupied by one conserved water molecule (W1), which also stabilizes that pocket. This position is occupied by a negatively charged atom of the substrate or ligand in ligand bound crystal structures. In fact, MD simulation study of Ser75 to Val indicates that W1 conserved water molecule is stabilized by Ser75, while Thr152, and His190 also act as anchor residues to maintain appropriate architecture of oxyanion pocket through water mediated H-bond interactions. Possibly, four conserved water molecules stabilize oxyanion hole in unliganded state, but they vacate these positions when the enzyme (hGMPS)-substrate complex is formed. Thus this study not only reveals functionally important role of conserved water molecules in GAT domain, but also highlights essential role of other non-catalytic residues such as Ser75 and Thr152 in this enzymatic domain. The results from this computational study could be of interest to experimental community and provide a testable hypothesis for experimental validation. Conserved sites of water molecules near and at oxyanion hole highlight structural importance of water molecules and suggest a rethink of the conventional definition of chemical geometry of inhibitor binding site. © 2016 Wiley Periodicals, Inc.

DOR/Tp53inp2 and Tp53inp1 constitute a metazoan gene family encoding dual regulators of autophagy and transcription.

PubMed

Sancho, Ana; Duran, Jordi; García-España, Antonio; Mauvezin, Caroline; Alemu, Endalkachew A; Lamark, Trond; Macias, Maria J; DeSalle, Rob; Royo, Miriam; Sala, David; Chicote, Javier U; Palacín, Manuel; Johansen, Terje; Zorzano, Antonio

2012-01-01

Human DOR/TP53INP2 displays a unique bifunctional role as a modulator of autophagy and gene transcription. However, the domains or regions of DOR that participate in those functions have not been identified. Here we have performed structure/function analyses of DOR guided by identification of conserved regions in the DOR gene family by phylogenetic reconstructions. We show that DOR is present in metazoan species. Invertebrates harbor only one gene, DOR/Tp53inp2, and in the common ancestor of vertebrates Tp53inp1 may have arisen by gene duplication. In keeping with these data, we show that human TP53INP1 regulates autophagy and that different DOR/TP53INP2 and TP53INP1 proteins display transcriptional activity. The use of molecular evolutionary information has been instrumental to determine the regions that participate in DOR functions. DOR and TP53INP1 proteins share two highly conserved regions (region 1, aa residues 28-42; region 2, 66-112 in human DOR). Mutation of conserved hydrophobic residues in region 1 of DOR (that are part of a nuclear export signal, NES) reduces transcriptional activity, and blocks nuclear exit and autophagic activity under autophagy-activated conditions. We also identify a functional and conserved LC3-interacting motif (LIR) in region 1 of DOR and TP53INP1 proteins. Mutation of conserved acidic residues in region 2 of DOR reduces transcriptional activity, impairs nuclear exit in response to autophagy activation, and disrupts autophagy. Taken together, our data reveal DOR and TP53INP1 as dual regulators of transcription and autophagy, and identify two conserved regions in the DOR family that concentrate multiple functions crucial for autophagy and transcription.

DOR/Tp53inp2 and Tp53inp1 Constitute a Metazoan Gene Family Encoding Dual Regulators of Autophagy and Transcription

PubMed Central

Sancho, Ana; Duran, Jordi; García-España, Antonio; Mauvezin, Caroline; Alemu, Endalkachew A.; Lamark, Trond; Macias, Maria J.; DeSalle, Rob; Royo, Miriam; Sala, David; Chicote, Javier U.; Palacín, Manuel; Johansen, Terje; Zorzano, Antonio

2012-01-01

Human DOR/TP53INP2 displays a unique bifunctional role as a modulator of autophagy and gene transcription. However, the domains or regions of DOR that participate in those functions have not been identified. Here we have performed structure/function analyses of DOR guided by identification of conserved regions in the DOR gene family by phylogenetic reconstructions. We show that DOR is present in metazoan species. Invertebrates harbor only one gene, DOR/Tp53inp2, and in the common ancestor of vertebrates Tp53inp1 may have arisen by gene duplication. In keeping with these data, we show that human TP53INP1 regulates autophagy and that different DOR/TP53INP2 and TP53INP1 proteins display transcriptional activity. The use of molecular evolutionary information has been instrumental to determine the regions that participate in DOR functions. DOR and TP53INP1 proteins share two highly conserved regions (region 1, aa residues 28–42; region 2, 66–112 in human DOR). Mutation of conserved hydrophobic residues in region 1 of DOR (that are part of a nuclear export signal, NES) reduces transcriptional activity, and blocks nuclear exit and autophagic activity under autophagy-activated conditions. We also identify a functional and conserved LC3-interacting motif (LIR) in region 1 of DOR and TP53INP1 proteins. Mutation of conserved acidic residues in region 2 of DOR reduces transcriptional activity, impairs nuclear exit in response to autophagy activation, and disrupts autophagy. Taken together, our data reveal DOR and TP53INP1 as dual regulators of transcription and autophagy, and identify two conserved regions in the DOR family that concentrate multiple functions crucial for autophagy and transcription. PMID:22470510

Correction: β-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade.

PubMed

Ito, Ryousuke; Nakada, Chika; Hoshino, Tsutomu

2017-01-18

Correction for 'β-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade' by Ryousuke Ito et al., Org. Biomol. Chem., 2017, 15, 177-188.

Experimental Investigation of Principal Residual Stress and Fatigue Performance for Turned Nickel-Based Superalloy Inconel 718.

PubMed

Hua, Yang; Liu, Zhanqiang

2018-05-24

Residual stresses of turned Inconel 718 surface along its axial and circumferential directions affect the fatigue performance of machined components. However, it has not been clear that the axial and circumferential directions are the principle residual stress direction. The direction of the maximum principal residual stress is crucial for the machined component service life. The present work aims to focuses on determining the direction and magnitude of principal residual stress and investigating its influence on fatigue performance of turned Inconel 718. The turning experimental results show that the principal residual stress magnitude is much higher than surface residual stress. In addition, both the principal residual stress and surface residual stress increase significantly as the feed rate increases. The fatigue test results show that the direction of the maximum principal residual stress increased by 7.4%, while the fatigue life decreased by 39.4%. The maximum principal residual stress magnitude diminished by 17.9%, whereas the fatigue life increased by 83.6%. The maximum principal residual stress has a preponderant influence on fatigue performance as compared to the surface residual stress. The maximum principal residual stress can be considered as a prime indicator for evaluation of the residual stress influence on fatigue performance of turned Inconel 718.

Identification of Novel 14-3-3 Residues That Are Critical for Isoform-specific Interaction with GluN2C to Regulate N-Methyl-d-aspartate (NMDA) Receptor Trafficking*

PubMed Central

Chung, Connie; Wu, Wei-Hua; Chen, Bo-Shiun

2015-01-01

The 14-3-3 family of proteins is widely distributed in the CNS where they are major regulators of essential neuronal functions. There are seven known mammalian 14-3-3 isoforms (ζ,, τ, ϵ, η, β, and σ), which generally function as adaptor proteins. Previously, we have demonstrated that 14-3-3ϵ isoform dynamically regulates forward trafficking of GluN2C-containing NMDA receptors (NMDARs) in cerebellar granule neurons, that when expressed on the surface, promotes neuronal survival following NMDA-induced excitotoxicity. Here, we report 14-3-3 isoform-specific binding and functional regulation of GluN2C. In particular, we show that GluN2C C-terminal domain (CTD) binds to all 14-3-3 isoforms except 14-3-3σ, and binding is dependent on GluN2C serine 1096 phosphorylation. Co-expression of 14-3-3 (ζ and ϵ) and GluN1/GluN2C promotes the forward delivery of receptors to the cell surface. We further identify novel residues serine 145, tyrosine 178, and cysteine 189 on α-helices 6, 7, and 8, respectively, within ζ-isoform as part of the GluN2C binding motif and independent of the canonical peptide binding groove. Mutation of these conserved residues abolishes GluN2C binding and has no functional effect on GluN2C trafficking. Reciprocal mutation of alanine 145, histidine 180, and isoleucine 191 on 14-3-3σ isoform promotes GluN2C binding and surface expression. Moreover, inhibiting endogenous 14-3-3 using a high-affinity peptide inhibitor, difopein, greatly diminishes GluN2C surface expression. Together, these findings highlight the isoform-specific structural and functional differences within the 14-3-3 family of proteins, which determine GluN2C binding and its essential role in targeting the receptor to the cell surface to facilitate glutamatergic neurotransmission. PMID:26229101

A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque.

PubMed

Terauchi, Takashi; Terashima, Hiroyuki; Kojima, Seiji; Homma, Michio

2011-08-01

Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion (H(+) or Na(+)) as their energy source, and are composed of stator and rotor proteins. Sodium-driven and proton-driven motors have the stator proteins PomA and PomB or MotA and MotB, respectively, which interact with each other in their transmembrane (TM) regions to form an ion channel. The single TM region of PomB or MotB, which forms the ion-conduction pathway together with TM3 and TM4 of PomA or MotA, respectively, has a highly conserved aspartate residue that is the ion binding site and is essential for rotation. To investigate the ion conductivity and selectivity of the Na(+)-driven PomA/PomB stator complex, we replaced conserved residues predicted to be near the conserved aspartate with H(+)-type residues, PomA-N194Y, PomB-F22Y and/or PomB-S27T. Motility analysis revealed that the ion specificity was not changed by either of the PomB mutations. PomB-F22Y required a higher concentration of Na(+) to exhibit swimming, but this effect was suppressed by additional mutations, PomA-N194Y or PomB-S27T. Moreover, the motility of the PomB-F22Y mutant was resistant to phenamil, a specific inhibitor for the Na(+) channel. When PomB-F22 was changed to other amino acids and the effects on swimming ability were investigated, replacement with a hydrophilic residue decreased the maximum swimming speed and conferred strong resistance to phenamil. From these results, we speculate that the Na(+) flux is reduced by the PomB-F22Y mutation, and that PomB-F22 is important for the effective release of Na(+) from PomB-D24.

Molecular Characterization of the Skate Peripherin/rds Gene: Relationship to Its Orthologues and Paralogues

PubMed Central

Li, Chibo; Ding, Xi-Qin; O’Brien, John; Al-Ubaidi, Muayyad R.

2010-01-01

PURPOSE A great deal of information about functionally significant domains of a protein may be obtained by comparison of primary sequences of gene homologues over a broad phylogenetic base. This study was designed to identify evolutionarily conserved domains of the photoreceptor disc membrane protein peripherin/rds by analysis of the homologue in a primitive vertebrate, the skate. METHODS A skate retinal cDNA library was screened using a mouse peripherin/rds clone. The 5′ and 3′ untranslated regions of the skate peripherin/rds (srds) cDNA were isolated by the rapid amplification of cDNA ends (RACE) approach. The gene structure was characterized by PCR amplification and sequencing of genomic fragments. Northern and Western blot analyses were used to identify srds transcript and protein, respectively. RESULTS A new homologue of peripherin/rds was identified from the skate retinal cDNA library. SRDS is a glycoprotein with a predicted molecular mass of 40.2 kDa. The srds gene consists of two exons and one small intron and transcribes into a single 6-kb message. Phylogenetic analysis places SRDS at the base of peripherin/rds family and near the division of that group and the branch leading to rds-like and rom-1 genes. SRDS protein is 54.5% identical with peripherin/rds across species. Identity is significantly higher (73%) in the intradiscal domains. Sequence comparison revealed the conservation of all residues that have been shown, on mutation, to associate with retinitis pigmentosa and showed conservation of most residues associated with macular dystrophies. Comparison with ROM-1 and other rds-like proteins revealed the presence of a highly conserved domain in the large intradiscal loop. CONCLUSIONS Srds represents the skate orthologue of mammalian peripherin/rds genes. Conservation of most of the residues associated with human retinal diseases indicates that these residues serve important functional roles. The high degree of conservation of a short stretch within the large intradiscal loop also suggests an important function for this domain. PMID:12766040

A cool approach to reducing electrode-induced trauma: Localized therapeutic hypothermia conserves residual hearing in cochlear implantation.

PubMed

Tamames, Ilmar; King, Curtis; Bas, Esperanza; Dietrich, W Dalton; Telischi, Fred; Rajguru, Suhrud M

2016-09-01

The trauma caused during cochlear implant insertion can lead to cell death and a loss of residual hair cells in the cochlea. Various therapeutic approaches have been studied to prevent cochlear implant-induced residual hearing loss with limited success. In the present study, we show the efficacy of mild to moderate therapeutic hypothermia of 4 to 6 °C applied to the cochlea in reducing residual hearing loss associated with the electrode insertion trauma. Rats were randomly distributed in three groups: control contralateral cochleae, normothermic implanted cochleae and hypothermic implanted cochleae. Localized hypothermia was delivered to the middle turn of the cochlea for 20 min before and after implantation using a custom-designed probe perfused with cooled fluorocarbon. Auditory brainstem responses (ABRs) were recorded to assess the hearing function prior to and post-cochlear implantation at various time points up to 30 days. At the conclusion of the trials, inner ears were harvested for histology and cell count. The approach was extended to cadaver temporal bones to study the potential surgical approach and efficacy of our device. In this case, the hypothermia probe was placed next to the round window niche via the facial recess or a myringotomy. A significant loss of residual hearing was observed in the normothermic implant group. Comparatively, the residual hearing in the cochleae receiving therapeutic hypothermia was significantly conserved. Histology confirmed a significant loss of outer hair cells in normothermic cochleae receiving the surgical trauma when compared to the hypothermia treated group. In human temporal bones, a controlled and effective cooling of the cochlea was achieved using our approach. Collectively, these results suggest that therapeutic hypothermia during cochlear implantation may reduce traumatic effects of electrode insertion and improve conservation of residual hearing. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A feature-based approach to modeling protein–protein interaction hot spots

PubMed Central

Cho, Kyu-il; Kim, Dongsup; Lee, Doheon

2009-01-01

Identifying features that effectively represent the energetic contribution of an individual interface residue to the interactions between proteins remains problematic. Here, we present several new features and show that they are more effective than conventional features. By combining the proposed features with conventional features, we develop a predictive model for interaction hot spots. Initially, 54 multifaceted features, composed of different levels of information including structure, sequence and molecular interaction information, are quantified. Then, to identify the best subset of features for predicting hot spots, feature selection is performed using a decision tree. Based on the selected features, a predictive model for hot spots is created using support vector machine (SVM) and tested on an independent test set. Our model shows better overall predictive accuracy than previous methods such as the alanine scanning methods Robetta and FOLDEF, and the knowledge-based method KFC. Subsequent analysis yields several findings about hot spots. As expected, hot spots have a larger relative surface area burial and are more hydrophobic than other residues. Unexpectedly, however, residue conservation displays a rather complicated tendency depending on the types of protein complexes, indicating that this feature is not good for identifying hot spots. Of the selected features, the weighted atomic packing density, relative surface area burial and weighted hydrophobicity are the top 3, with the weighted atomic packing density proving to be the most effective feature for predicting hot spots. Notably, we find that hot spots are closely related to π–related interactions, especially π · · · π interactions. PMID:19273533

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

PubMed

Keyamura, Kenji; Katayama, Tsutomu

2011-08-19

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

PubMed Central

Keyamura, Kenji; Katayama, Tsutomu

2011-01-01

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

Fall conservation deep tillage stabilizes maize residues into soil organic matter

USDA-ARS?s Scientific Manuscript database

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...

Crystal structures of two novel dye-decolorizing peroxidases reveal a beta-barrel fold with a conserved heme-binding motif.

PubMed

Zubieta, Chloe; Krishna, S Sri; Kapoor, Mili; Kozbial, Piotr; McMullan, Daniel; Axelrod, Herbert L; Miller, Mitchell D; Abdubek, Polat; Ambing, Eileen; Astakhova, Tamara; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C; Duan, Lian; Elsliger, Marc-André; Feuerhelm, Julie; Grzechnik, Slawomir K; Hale, Joanna; Hampton, Eric; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kumar, Abhinav; Marciano, David; Morse, Andrew T; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Reyes, Ron; Rife, Christopher L; Schimmel, Paul; van den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Xu, Qingping; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2007-11-01

BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 A, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, alpha+beta ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein). (c) 2007 Wiley-Liss, Inc.

Balancing energy, conservation, and soil health requirements for plant biomass

USDA-ARS?s Scientific Manuscript database

The importance of crop residue for mitigating water and wind erosion, sustaining soil organic carbon (SOC) levels, and providing animal feed and bedding is well recognized around the world. But, those critical needs are no longer the only factors influencing crop residue management decisions. Global...

Properties of the [NiFe]-hydrogenase maturation protein HypD.

PubMed

Blokesch, Melanie; Böck, August

2006-07-24

A mutational screen of amino acid residues of hydrogenase maturation protein HypD from Escherichia coli disclosed that seven conserved cysteine residues located in three different motifs in HypD are essential. Evidence is presented for potential functions of these motifs in the maturation process.

Termination of cover crops using rollers/crimpers

USDA-ARS?s Scientific Manuscript database

An integral component of conservation agriculture systems is the use of a high-residue winter cover crop; however, terminating cover crops is an addition expense and planting into high-residue can be a challenge. An experiment was conducted using black oat (Avena strigosa Schreb.), rye (Secale cere...

Coevolving residues of (beta/alpha)(8)-barrel proteins play roles in stabilizing active site architecture and coordinating protein dynamics.

PubMed

Shen, Hongbo; Xu, Feng; Hu, Hairong; Wang, Feifei; Wu, Qi; Huang, Qiang; Wang, Honghai

2008-12-01

Indole-3-glycerol phosphate synthase (IGPS) is a representative of (beta/alpha)(8)-barrel proteins-the most common enzyme fold in nature. To better understand how the constituent amino-acids work together to define the structure and to facilitate the function, we investigated the evolutionary and dynamical coupling of IGPS residues by combining statistical coupling analysis (SCA) and molecular dynamics (MD) simulations. The coevolving residues identified by the SCA were found to form a network which encloses the active site completely. The MD simulations showed that these coevolving residues are involved in the correlated and anti-correlated motions. The correlated residues are within van der Waals contact and appear to maintain the active site architecture; the anti-correlated residues are mainly distributed on opposite sides of the catalytic cavity and coordinate the motions likely required for the substrate entry and product release. Our findings might have broad implications for proteins with the highly conserved (betaalpha)(8)-barrel in assessing the roles of amino-acids that are moderately conserved and not directly involved in the active site of the (beta/alpha)(8)-barrel. The results of this study could also provide useful information for further exploring the specific residue motions for the catalysis and protein design based on the (beta/alpha)(8)-barrel scaffold.

A slender tract of glycine residues is required for translocation of the VP2 protein N-terminal domain through the parvovirus MVM capsid channel to initiate infection.

PubMed

Castellanos, Milagros; Pérez, Rebeca; Rodríguez-Huete, Alicia; Grueso, Esther; Almendral, José M; Mateu, Mauricio G

2013-10-01

Viruses constitute paradigms to study conformational dynamics in biomacromolecular assemblies. Infection by the parvovirus MVM (minute virus of mice) requires a conformational rearrangement that involves the intracellular externalization through capsid channels of the 2Nt (N-terminal region of VP2). We have investigated the role in this process of conserved glycine residues in an extended glycine-rich tract located immediately after 2Nt. Based on the virus structure, residues with hydrophobic side chains of increasing volume were substituted for glycine residues 31 or 33. Mutations had no effect on capsid assembly or stability, but inhibited virus infectivity. All mutations, except those to alanine residues which had minor effects, impaired 2Nt externalization in nuclear maturing virions and in purified virions, to an extent that correlated with the side chain size. Different biochemical and biophysical analyses were consistent with this result. Importantly, all of the tested glycine residue replacements impaired the capacity of the virion to initiate infection, at ratios correlating with their restrictive effects on 2Nt externalization. Thus small residues within the evolutionarily conserved glycine-rich tract facilitate 2Nt externalization through the capsid channel, as required by this virus to initiate cell entry. The results demonstrate the exquisite dependence on geometric constraints of a biologically relevant translocation event in a biomolecular complex.

Low-stringency selection of TEM1 for BLIP shows interface plasticity and selection for faster binders

PubMed Central

Cohen-Khait, Ruth; Schreiber, Gideon

2016-01-01

Protein–protein interactions occur via well-defined interfaces on the protein surface. Whereas the location of homologous interfaces is conserved, their composition varies, suggesting that multiple solutions may support high-affinity binding. In this study, we examined the plasticity of the interface of TEM1 β-lactamase with its protein inhibitor BLIP by low-stringency selection of a random TEM1 library using yeast surface display. Our results show that most interfacial residues could be mutated without a loss in binding affinity, protein stability, or enzymatic activity, suggesting plasticity in the interface composition supporting high-affinity binding. Interestingly, many of the selected mutations promoted faster association. Further selection for faster binders was achieved by drastically decreasing the library–ligand incubation time to 30 s. Preequilibrium selection as suggested here is a novel methodology for specifically selecting faster-associating protein complexes. PMID:27956635

The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion.

PubMed Central

Ringwald, M; Schuh, R; Vestweber, D; Eistetter, H; Lottspeich, F; Engel, J; Dölz, R; Jähnig, F; Epplen, J; Mayer, S

1987-01-01

We have determined the amino acid sequence of the Ca2+-dependent cell adhesion molecule uvomorulin as it appears on the cell surface. The extracellular part of the molecule exhibits three internally repeated domains of 112 residues which are most likely generated by gene duplication. Each of the repeated domains contains two highly conserved units which could represent putative Ca2+-binding sites. Secondary structure predictions suggest that the putative Ca2+-binding units are located in external loops at the surface of the protein. The protein sequence exhibits a single membrane-spanning region and a cytoplasmic domain. Sequence comparison reveals extensive homology to the chicken L-CAM. Both uvomorulin and L-CAM are identical in 65% of their entire amino acid sequence suggesting a common origin for both CAMs. Images Fig. 1. Fig. 4. Fig. 7. PMID:3501370

Methylated glycans as conserved targets of animal and fungal innate defense

PubMed Central

Wohlschlager, Therese; Butschi, Alex; Grassi, Paola; Sutov, Grigorij; Gauss, Robert; Hauck, Dirk; Schmieder, Stefanie S.; Knobel, Martin; Titz, Alexander; Dell, Anne; Haslam, Stuart M.; Hengartner, Michael O.; Aebi, Markus; Künzler, Markus

2014-01-01

Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of β-propeller lectins conserved from bacteria to mammals that have been shown to bind bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans, suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1, coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus, showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor. PMID:24879441

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

PubMed Central

Teriete, Peter; Thai, Khang; Choi, Jungyuen; Marassi, Francesca M.

2009-01-01

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na+/K+ ion binding site of the Na,K-ATPase α subunit. PMID:19761758

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

StralSV: assessment of sequence variability within similar 3D structures and application to polio RNA-dependent RNA polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zemla, A; Lang, D; Kostova, T

2010-11-29

Most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. A serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the low confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. Multiple sequence alignment methods are more satisfactory - still, they cannot provide reliable results at low levels of sequence identity. Our goal in the current work was to develop an algorithm that could overcome these difficulties and facilitatemore » the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. Here we present StralSV, a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. We apply StralSV in a study of the RNA-dependent RNA polymerase of poliovirus and demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique or that shared structural similarity with structures that are distantly related. By quantifying residue frequencies among many residue-residue pairs extracted from local alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. We further demonstrate that considerable detailed structural and phylogenetic information can be derived from StralSV analyses. StralSV is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. StralSV analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected residues at a given sequence position.« less

StralSV: assessment of sequence variability within similar 3D structures and application to polio RNA-dependent RNA polymerase.

PubMed

Zemla, Adam T; Lang, Dorothy M; Kostova, Tanya; Andino, Raul; Ecale Zhou, Carol L

2011-06-02

Most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. A serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the low confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. Multiple sequence alignment methods are more satisfactory--still, they cannot provide reliable results at low levels of sequence identity. Our goal in the current work was to develop an algorithm that could help overcome these difficulties by facilitating the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. Here we present StralSV (structure-alignment sequence variability), a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. We apply StralSV in a study of the RNA-dependent RNA polymerase of poliovirus, and we demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique, or that share structural similarity with proteins that would be considered distantly related. By quantifying residue frequencies among many residue-residue pairs extracted from local structural alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. We further demonstrate that considerable detailed structural and phylogenetic information can be derived from StralSV analyses. StralSV is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. StralSV analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected residues at a given sequence position. StralSV is provided as a web service at http://proteinmodel.org/AS2TS/STRALSV/.

Research on Formation Mechanism of Dynamic Response and Residual Stress of Sheet Metal Induced by Laser Shock Wave

NASA Astrophysics Data System (ADS)

Feng, Aixin; Cao, Yupeng; Wang, Heng; Zhang, Zhengang

2018-01-01

In order to reveal the quantitative control of the residual stress on the surface of metal materials, the relevant theoretical and experimental studies were carried out to investigate the dynamic response of metal thin plates and the formation mechanism of residual stress induced by laser shock wave. In this paper, the latest research trends on the surface residual stress of laser shock processing technology were elaborated. The main progress of laser shock wave propagation mechanism and dynamic response, laser shock, and surface residual stress were discussed. It is pointed out that the multi-scale characterization of laser and material, surface residual stress and microstructure change is a new hotspot in laser shock strengthening technology.

Valid internal standard technique for arson detection based on gas chromatography-mass spectrometry.

PubMed

Salgueiro, Pedro A S; Borges, Carlos M F; Bettencourt da Silva, Ricardo J N

2012-09-28

The most popular procedures for the detection of residues of accelerants in fire debris are the ones published by the American Society for Testing and Materials (ASTM E1412-07 and E1618-10). The most critical stages of these tests are the conservation of fire debris from the sampling to the laboratory, the extraction of residues of accelerants from the debris to the activated charcoal strips (ACS) and from those to the final solvent, as well as the analysis of sample extract by gas chromatography-mass spectrometry (GC-MS) and the interpretation of the instrumental signal. This work proposes a strategy for checking the quality of the sample conservation, the accelerant residues transference to final solvent and GC-MS analysis, using internal standard additions. It is used internal standards ranging from a highly volatile compound for checking debris conservation to low volatile compound for checking GC-MS repeatability. The developed quality control (QC) parameters are not affected by GC-MS sensitivity variation and, specifically, the GC-MS performance control is not affected by ACS adsorption saturation that may mask test performance deviations. The proposed QC procedure proved to be adequate to check GC-MS repeatability, ACS extraction and sample conservation since: (1) standard additions are affected by negligible uncertainty and (2) observed dispersion of QC parameters are fit for its intended use. Copyright © 2012 Elsevier B.V. All rights reserved.

Analysis of nucleotide diphosphate sugar dehydrogenases reveals family and group-specific relationships.

PubMed

Freas, Nicholas; Newton, Peter; Perozich, John

2016-01-01

UDP-glucose dehydrogenase (UDPGDH), UDP-N-acetyl-mannosamine dehydrogenase (UDPNAMDH) and GDP-mannose dehydrogenase (GDPMDH) belong to a family of NAD (+)-linked 4-electron-transfering oxidoreductases called nucleotide diphosphate sugar dehydrogenases (NDP-SDHs). UDPGDH is an enzyme responsible for converting UDP-d-glucose to UDP-d-glucuronic acid, a product that has different roles depending on the organism in which it is found. UDPNAMDH and GDPMDH convert UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid and GDP-mannose to GDP-mannuronic acid, respectively, by a similar mechanism to UDPGDH. Their products are used as essential building blocks for the exopolysaccharides found in organisms like Pseudomonas aeruginosa and Staphylococcus aureus. Few studies have investigated the relationships between these enzymes. This study reveals the relationships between the three enzymes by analysing 229 amino acid sequences. Eighteen invariant and several other highly conserved residues were identified, each serving critical roles in maintaining enzyme structure, coenzyme binding or catalytic function. Also, 10 conserved motifs that included most of the conserved residues were identified and their roles proposed. A phylogenetic tree demonstrated relationships between each group and verified group assignment. Finally, group entropy analysis identified novel conservations unique to each NDP-SDH group, including residue positions critical to NDP-sugar substrate interaction, enzyme structure and intersubunit contact. These positions may serve as targets for future research. UDP-glucose dehydrogenase (UDPGDH, EC 1.1.1.22).

Additional Surgery after Breast-Conserving Surgery Varies Widely

Cancer.gov

A study published in the Feb. 1, 2012, issue of JAMA found that the number of women who have one or more additional surgeries to remove suspected residual tumor tissue (re-excisions) following breast-conserving surgery (BCS) for breast cancer varies widely across surgeons and hospitals.

Crop rotation and residue management effects on deficit irrigated cotton and corn

USDA-ARS?s Scientific Manuscript database

Compared with disk tillage (DT), no-tillage (NT) retains residue and increases precipitation capture, which has the potential to reduce irrigation withdrawals from the declining Ogallala Aquifer. Our objective was to quantify DT and NT effects on water conservation during fallow and the subsequent y...

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.

Crystal structure of a papain-fold protein without the catalytic residue: a novel member in the cysteine proteinase family.

PubMed

Zhang, Min; Wei, Zhiyi; Chang, Shaojie; Teng, Maikun; Gong, Weimin

2006-04-21

A 31kDa cysteine protease, SPE31, was isolated from the seeds of a legume plant, Pachyrizhus erosus. The protein was purified, crystallized and the 3D structure solved using molecular replacement. The cDNA was obtained by RT PCR followed by amplification using mRNA isolated from the seeds of the legume plant as a template. Analysis of the cDNA sequence and the 3D structure indicated the protein to belong to the papain family. Detailed analysis of the structure revealed an unusual replacement of the conserved catalytic Cys with Gly. Replacement of another conserved residue Ala/Gly by a Phe sterically blocks the access of the substrate to the active site. A polyethyleneglycol molecule and a natural peptide fragment were bound to the surface of the active site. Asn159 was found to be glycosylated. The SPE31 cDNA sequence shares several features with P34, a protein found in soybeans, that is implicated in plant defense mechanisms as an elicitor receptor binding to syringolide. P34 has also been shown to interact with vegetative storage proteins and NADH-dependent hydroxypyruvate reductase. These roles suggest that SPE31 and P34 form a unique subfamily within the papain family. The crystal structure of SPE31 complexed with a natural peptide ligand reveals a unique active site architecture. In addition, the clear evidence of glycosylated Asn159 provides useful information towards understanding the functional mechanism of SPE31/P34.

Species-specific functional evolution of neuroglobin.

PubMed

Wakasugi, Keisuke; Takahashi, Nozomu; Uchida, Hiroyuki; Watanabe, Seiji

2011-09-01

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Human Ngb is involved in neuroprotection under oxidative stress conditions such as ischemia and reperfusion. We previously demonstrated that, on the one hand, human ferric Ngb binds to the α-subunit of heterotrimeric G proteins (Gα(i)) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Gα(i). On the other hand, zebrafish Ngb does not exhibit GDI activity. By using wild-type and Ngb mutants, we demonstrated that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. The crucial residues for both GDI and neuroprotective activity, corresponding to Glu53, Arg97, Glu118, and Glu151 of human Ngb, are conserved among boreotheria of mammalia. Recently, we found that zebrafish, but not human, Ngb can translocate into cells and clarified that module M1 of zebrafish Ngb is important for protein transduction. By performing site-directed mutagenesis, we showed that Lys7, Lys9, Lys21, and Lys23 of zebrafish Ngb are crucial for protein transduction activity. Because these residues are conserved among fishes, but not among mammals, birds, reptilians, or amphibians, the ability to penetrate cell membranes may be a unique characteristic of fish Ngb proteins. Moreover, we clarified that zebrafish Ngb interacts with negatively charged cell-surface glycosaminoglycan. Taken together, these results suggest that the function of Ngb proteins has been changing dynamically throughout the evolution of life. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of Turning and Ball Burnishing on the Microstructure and Residual Stress Distribution in Stainless Steel Cold Spray Deposits

NASA Astrophysics Data System (ADS)

Sova, A.; Courbon, C.; Valiorgue, F.; Rech, J.; Bertrand, Ph.

2017-12-01

In this paper, an experimental study of influence of machining by turning and ball burnishing on the surface morphology, structure and residual stress distribution of cold spray 17-4 PH stainless steel deposits is provided. It is shown that cold spray deposits could be machined by turning under parameters closed to turning of bulk 17-4 PH stainless steel. Ball burnishing process permits to decrease surface roughness. Cross-sectional observation revealed that the turning and ball burnishing process allowed microstructure changes in the coating near-surface zone. In particular, significant particle deformation and particle boundary fragmentation is observed. Measurements of residual stresses showed that residual stresses in the as-spray deposit are compressive. After machining by turning, tensile residual stresses in the near-surface zone were induced. Further surface finishing of turned coating by ball burnishing allowed the establishment of the compressive residual stresses in the coating.

The primary structure of stinging nettle (Urtica dioica) agglutinin. A two-domain member of the hevein family.

PubMed

Beintema, J J; Peumans, W J

1992-03-09

The primary structure of stinging nettle (Urtica dioica) agglutinin has been determined by sequence analysis of peptides obtained from three overlapping proteolytic digests. The sequence of 80 residues consists of two hevein-like domains with the same spacing of half-cystine residues and several other conserved residues as observed earlier in other proteins with hevein-like domains. The hinge region between the two domains is four residues longer than those between the four domains in cereal lectins like wheat germ agglutinin.

Evolutionarily conserved regions and hydrophobic contacts at the superfamily level: The case of the fold-type I, pyridoxal-5′-phosphate-dependent enzymes

PubMed Central

Paiardini, Alessandro; Bossa, Francesco; Pascarella, Stefano

2004-01-01

The wealth of biological information provided by structural and genomic projects opens new prospects of understanding life and evolution at the molecular level. In this work, it is shown how computational approaches can be exploited to pinpoint protein structural features that remain invariant upon long evolutionary periods in the fold-type I, PLP-dependent enzymes. A nonredundant set of 23 superposed crystallographic structures belonging to this superfamily was built. Members of this family typically display high-structural conservation despite low-sequence identity. For each structure, a multiple-sequence alignment of orthologous sequences was obtained, and the 23 alignments were merged using the structural information to obtain a comprehensive multiple alignment of 921 sequences of fold-type I enzymes. The structurally conserved regions (SCRs), the evolutionarily conserved residues, and the conserved hydrophobic contacts (CHCs) were extracted from this data set, using both sequence and structural information. The results of this study identified a structural pattern of hydrophobic contacts shared by all of the superfamily members of fold-type I enzymes and involved in native interactions. This profile highlights the presence of a nucleus for this fold, in which residues participating in the most conserved native interactions exhibit preferential evolutionary conservation, that correlates significantly (r = 0.70) with the extent of mean hydrophobic contact value of their apolar fraction. PMID:15498941

A conserved tryptophan within the WRDPLVDID domain of yeast Pah1 phosphatidate phosphatase is required for its in vivo function in lipid metabolism.

PubMed

Park, Yeonhee; Han, Gil-Soo; Carman, George M

2017-12-01

PAH1 -encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol at the endoplasmic reticulum membrane, plays a major role in controlling the utilization of phosphatidate for the synthesis of triacylglycerol or membrane phospholipids. The conserved N-LIP and haloacid dehalogenase-like domains of Pah1 are required for phosphatidate phosphatase activity and the in vivo function of the enzyme. Its non-conserved regions, which are located between the conserved domains and at the C terminus, contain sites for phosphorylation by multiple protein kinases. Truncation analyses of the non-conserved regions showed that they are not essential for the catalytic activity of Pah1 and its physiological functions ( e.g. triacylglycerol synthesis). This analysis also revealed that the C-terminal region contains a previously unrecognized WRDPLVDID domain (residues 637-645) that is conserved in yeast, mice, and humans. The deletion of this domain had no effect on the catalytic activity of Pah1 but caused the loss of its in vivo function. Site-specific mutational analyses of the conserved residues within WRDPLVDID indicated that Trp-637 plays a crucial role in Pah1 function. This work also demonstrated that the catalytic activity of Pah1 is required but is not sufficient for its in vivo functions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

High throughput secondary electron imaging of organic residues on a graphene surface

NASA Astrophysics Data System (ADS)

Zhou, Yangbo; O'Connell, Robert; Maguire, Pierce; Zhang, Hongzhou

2014-11-01

Surface organic residues inhibit the extraordinary electronic properties of graphene, hindering the development of graphene electronics. However, fundamental understanding of the residue morphology is still absent due to a lack of high-throughput and high-resolution surface characterization methods. Here, we demonstrate that secondary electron (SE) imaging in the scanning electron microscope (SEM) and helium ion microscope (HIM) can provide sub-nanometer information of a graphene surface and reveal the morphology of surface contaminants. Nanoscale polymethyl methacrylate (PMMA) residues are visible in the SE imaging, but their contrast, i.e. the apparent lateral dimension, varies with the imaging conditions. We have demonstrated a quantitative approach to readily obtain the physical size of the surface features regardless of the contrast variation. The fidelity of SE imaging is ultimately determined by the probe size of the primary beam. HIM is thus evaluated to be a superior SE imaging technique in terms of surface sensitivity and image fidelity. A highly efficient method to reveal the residues on a graphene surface has therefore been established.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Li; DeRider, Michele; McCornack, Milissa A.

Chemokines (chemotactic cytokines) comprise a large family of proteins that recruit and activate leukocytes, giving chemokines a major role in both the immune response and inflammation-related diseases. The poxvirus-encoded viral CC chemokine inhibitor (vCCI) binds to many CC chemokines with high affinity, acting as a potent inhibitor of chemokine action. We have used heteronuclear multidimensional NMR to determine the first structure of an orthopoxvirus vCCI in complex with a human CC chemokine MIP-1β. vCCI binds to the chemokine with 1:1 stoichiometry, using residues from its β-sheet II to interact with the a surface of MIP-1β that includes the N-terminus, themore » following residues in the so-called N-loop20’s region, and the 40’s loop. This structure reveals a general strategy of vCCI for selective chemokine binding, as vCCI appears to interact most stronglyinteracts most directly with residues that are conserved among a subset of CC chemokines, but are not conservednot among the other chemokine subfamilies. This structure reveals a general strategy of vCCI for selective chemokine binding. Chemokines play critical roles in the immune system, causing chemotaxis of a variety of cells to sites of infection and inflammation, as well as mediating cell homing and immune system development 1(Baggiolini 2001). To date, about 50 chemokines have been identified, and these small proteins (7-14 kDa) are believed to function by binding with endothelial or matrix glycosaminoglycans to form a concentration gradient that is then sensed by high affinity, 7-transmembrane domain G-protein coupled chemokine receptors on the surface of immune cells surface. The chemokine system is critical for host defense in healthy individuals, butand can also lead to diseases including asthma, arthritis, and atherosclerosis in the case of malfunction, often due to inappropriate inflammation and subsequent tissue damage 2(Gerard and Rollins 2001). There are four subfamilies of chemokines, CC, CXC, C, and CX3C, named for the position of conserved N-terminal cysteine residues. Members of the same subfamily often have overlapping receptor binding and cell activation ability while different subfamilies tend to functionwork on different cell subsets1{Baggiolini, 2001 #472} (REF). For example, CC chemokines mostly interact with monocytes, macrophages, T cells and eosinophils, while CXC chemokines mainly interact with neutrophils. Structures of chemokines from different subfamilies have been solved by NMR and X-ray crystallography (XXX Include Fernandez and Lolis review) 3-9{Clore, 1990 #91;Skelton, 1995 #97;Handel, 1996 #93;Crump, 1997 #92;Crump, 1998 #248;Meunier, 1997 #96;Fernandez, 2002 #496}(Clore 1990; Skelton 1995; Handel and Domaille 1996; Crump, Gong et al. 1997; Meunier 1997; Crump, Rajarathnam et al. 1998)(Clore 1990; Skelton 1995; Handel and Domaille 1996; Crump et al. 1997; Meunier 1997; Crump et al. 1998).« less

Conservation agriculture among small scale farmers in semi-arid region of Kenya does improve soil biological quality and soil organic carbon

NASA Astrophysics Data System (ADS)

Waweru, Geofrey; Okoba, Barrack; Cornelis, Wim

2016-04-01

The low food production in Sub-Saharan Africa (SSA) has been attributed to declining soil quality. This is due to soil degradation and fertility depletion resulting from unsustainable conventional farming practices such as continuous tillage, crop residue burning and mono cropping. To overcome these challenges, conservation agriculture (CA) is actively promoted. However, little has been done in evaluating the effect of each of the three principles of CA namely: minimum soil disturbance, maximum surface cover and diversified/crop rotation on soil quality in SSA. A study was conducted for three years from 2012 to 2015 in Laikipia East sub county in Kenya to evaluate the effect of tillage, surface cover and intercropping on a wide variety of physical, chemical and biological soil quality indicators, crop parameters and the field-water balance. This abstract reports on soil microbial biomass carbon (SMBC) and soil organic carbon (SOC). The experimental set up was a split plot design with tillage as main treatment (conventional till (CT), no-till (NT) and no-till with herbicide (NTH)), and intercropping and surface cover as sub treatment (intercropping maize with: beans, MB; beans and leucaena, MBL; beans and maize residues at 1.5 Mg ha-1 MBMu, and dolichos, MD). NT had significantly higher SMBC by 66 and 31% compared with CT and NTH respectively. SOC was significantly higher in NTH than CT and NT by 15 and 4%, respectively. Intercropping and mulching had significant effect on SMBC and SOC. MBMu resulted in higher SMBC by 31, 38 and 43%, and SOC by 9, 20 and 22% as compared with MBL, MD and MB, respectively. SMBC and SOC were significantly affected by the interaction between tillage, intercropping and soil cover with NTMBMu and NTHMBMu having the highest SMBC and SOC, respectively. We conclude that indeed tillage, intercropping and mulching substantially affect SMBC and SOC. On the individual components of CA, tillage and surface cover had the highest effect on SMBC and SOC, respectively, but the highest positive effect was realized when all the three principles were applied consecutively. Therefore, CA has the potential to improve biological soil quality among small scale rainfed farmers and thus promote sustainable production.

Impact of agronomic practices on soil biological properties on the Texas High Plains

USDA-ARS?s Scientific Manuscript database

Semiarid environments of the Texas High Plains decrease soil organic carbon and soil residue building resulting in difficult conditions for soil microbes. Current conservation practices such as conservation tillage, crop rotation, and cover cropping have not been quickly adopted in the area. In or...

The effects of mulching, tillage, and herbicides on weed control and watermelon yield

USDA-ARS?s Scientific Manuscript database

Currently few producers in the Southeast US have adopted conservation tillage practices in specialty crop production. The lack of conservation adoption is likely due to the added challenges in producing vegetables in cover crop residues, especially high biomass cover crop systems. The objective of t...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brandao, T.; Robinson, H; Johnson, S

Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presencemore » of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.« less

Dramatically reduced surface expression of NK cell receptor KIR2DS3 is attributed to multiple residues throughout the molecule.

PubMed

VandenBussche, C J; Mulrooney, T J; Frazier, W R; Dakshanamurthy, S; Hurley, C K

2009-03-01

Using flow cytometry, fluorescent microscopy and examination of receptor glycosylation status, we demonstrate that an entire killer cell immunoglobulin-like receptor (KIR) locus (KIR2DS3)--assumed earlier to be surface expressed--appears to have little appreciable surface expression in transfected cells. This phenotype was noted for receptors encoded by three allelic variants including the common KIR2DS3*001 allele. Comparing the surface expression of KIR2DS3 with that of the better-studied KIR2DS1 molecule in two different cell lines, mutational analysis identified multiple polymorphic amino-acid residues that significantly alter the proportion of molecules present on the cell surface. A simultaneous substitution of five residues localized to the leader peptide (residues -18 and -7), second domain (residues 123 and 150) and transmembrane region (residue 234) was required to restore KIR2DS3 to the expression level of KIR2DS1. Corresponding simultaneous substitutions of KIR2DS1 to the KIR2DS3 residues resulted in a dramatically decreased surface expression. Molecular modeling was used to predict how these substitutions contribute to this phenotype. Alterations in receptor surface expression are likely to affect the balance of immune cell signaling impacting the characteristics of the response to pathogens or malignancy.

Analysis of structural changes in active site of luciferase adsorbed on nanofabricated hydrophilic Si surface by molecular-dynamics simulations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishiyama, Katsuhiko; Hoshino, Tadatsugu; Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522

2007-05-21

Interactions between luciferase and a nanofabricated hydrophilic Si surface were explored by molecular-dynamics simulations. The structural changes in the active-site residues, the residues affecting the luciferin binding, and the residues affecting the bioluminescence color were smaller on the nanofabricated hydrophilic Si surface than on both a hydrophobic Si surface and a hydrophilic Si surface. The nanofabrication and wet-treatment techniques are expected to prevent the decrease in activity of luciferase on the Si surface.

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901.

PubMed

Campos-Olivas, R; Hörr, I; Bormann, C; Jung, G; Gronenborn, A M

2001-05-11

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction. Copyright 2001 Academic Press.

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

USDA-ARS?s Scientific Manuscript database

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...

Weed science and management, in soil sciences, land cover, and land use

USDA-ARS?s Scientific Manuscript database

An integral component of conservation agriculture systems in cotton is the use of a high-residue winter cover crop; however, terminating such cover crops is a cost and planting into high-residue is a challenge. Black oat, rye, and wheat winter cover crops were flattened with a straight-blade mechan...

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

USDA-ARS?s Scientific Manuscript database

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 ...

Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

USDA-ARS?s Scientific Manuscript database

In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

Evaluation of Surface Residual Stresses in Friction Stir Welds Due to Laser and Shot Peening

NASA Technical Reports Server (NTRS)

Hatamleh, Omar; Rivero, Iris V.; Lyons, Jed

2007-01-01

The effects of laser, and shot peening on the residual stresses in Friction Stir Welds (FSW) has been investigated. The surface residual stresses were measured at five different locations across the weld in order to produce an adequate residual stress profile. The residual stresses before and after sectioning the coupon from the welded plate were also measured, and the effect of coupon size on the residual stress relaxation was determined and characterized. Measurements indicate that residual stresses were not uniform along the welded plate, and large variation in stress magnitude could be exhibited at various locations along the FSW plate. Sectioning resulted in significant residual stress relaxation in the longitudinal direction attributed to the large change in dimensions in this direction. Overall, Laser and shot peening resulted in a significant reduction in tensile residual stresses at the surface of the specimens.

Structural basis for new pattern of conserved amino acid residues related to chitin-binding in the antifungal peptide from the coconut rhinoceros beetle Oryctes rhinoceros.

PubMed

Hemmi, Hikaru; Ishibashi, Jun; Tomie, Tetsuya; Yamakawa, Minoru

2003-06-20

Scarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros is a 36-residue polypeptide that has antifungal activity. The solution structure of scarabaecin has been determined from twodimensional 1H NMR spectroscopic data and hybrid distance geometry-simulated annealing protocol calculation. Based on 492 interproton and 10 hydrogen-bonding distance restraints and 36 dihedral angle restraints, we obtained 20 structures. The average backbone root-mean-square deviation for residues 4-35 is 0.728 +/- 0.217 A from the mean structure. The solution structure consists of a two-stranded antiparallel beta-sheet connected by a type-I beta-turn after a short helical turn. All secondary structures and a conserved disulfide bond are located in the C-terminal half of the peptide, residues 18-36. Overall folding is stabilized by a combination of a disulfide bond, seven hydrogen bonds, and numerous hydrophobic interactions. The structural motif of the C-terminal half shares a significant tertiary structural similarity with chitin-binding domains of plant and invertebrate chitin-binding proteins, even though scarabaecin has no overall sequence similarity to other peptide/polypeptides including chitin-binding proteins. The length of its primary structure, the number of disulfide bonds, and the pattern of conserved functional residues binding to chitin in scarabaecin differ from those of chitin-binding proteins in other invertebrates and plants, suggesting that scarabaecin does not share a common ancestor with them. These results are thought to provide further strong experimental evidence to the hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process.

Cloning, Site-Directed Mutagenesis, and Functional Analysis of Active Residues in Lymantria dispar Chitinase.

PubMed

Fan, Xiao-Jun; Yang, Chun; Zhang, Chang; Ren, Hui; Zhang, Jian-Dong

2018-01-01

Chitinases are glycosyl hydrolases that catalyze the hydrolysis of β-(1,4)-glycosidic bonds in chitin, the major structural polysaccharide presented in the cuticle and gut peritrophic matrix of insects. Two aspartate residues (D143, D145) and one tryptophan (W146) in the Lymantria dispar chitinase are highly conserved residues observed within the second conserved motif of the family 18 chitinase catalytic region. In this study, a chitinase cDNA, LdCht5, was cloned from L. dispar, and the roles of the three residues were investigated using site-directed mutagenesis and substituting them with three other amino acids. Seven mutant proteins, D143E, D145E, W146G, D143E/D145E, D143E/W146G, D145E/W146G, and D143E/D145E/W146G, as well as the wild-type enzyme, were produced using the baculovirus-insect cell line expression system. The enzymatic and kinetic properties of these mutant enzymes were measured using the oligosaccharide substrate MU-(GlcNAc) 3 . Among the seven mutants, the D145E, D143E/D145E, and D145E/W146G mutations kept some extant catalytic activity toward MU-(GlcNAc) 3 , while the D143E, W146G, D143E/W146G, and D143E/D145E/W146G mutant enzymes were inactivated. Compared with the mutant enzymes, the wild-type enzyme had higher values of k cat and k cat / K m . A study of the multiple point mutations in the second conserved catalytic region would help to elucidate the role of the critical residues and their relationships.

Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

NASA Astrophysics Data System (ADS)

Fleishman, Sarel

2012-02-01

Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

Northwest Energy Policy Project. Energy conservation policy evaluation: study module IA. Final report. Volume I. Summary report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berney, R.E.; Butcher, W.R.; Carter, L.F.

1977-01-01

This summary report identifies potential energy conservation measures and evaluates measures and policy options not likely to be suitable for adoption in the Pacific Northwest. Potential and incentives for energy conservation are specifically identified in the residential, commercial, industrial, and transportation sectors and through urban design. Selection of preferable policies and estimation of response to policies are next discussed. Finally, a computer impact model for calculating energy savings and changes in the levels of residuals resulting from energy conservation activities is discussed. (MCW)

Two residues in the basic region of the yeast transcription factor Yap8 are crucial for its DNA-binding specificity.

PubMed

Amaral, Catarina; Pimentel, Catarina; Matos, Rute G; Arraiano, Cecília M; Matzapetakis, Manolis; Rodrigues-Pousada, Claudina

2013-01-01

In Saccharomyces cerevisiae, the transcription factor Yap8 is a key determinant in arsenic stress response. Contrary to Yap1, another basic region-leucine zipper (bZIP) yeast regulator, Yap8 has a very restricted DNA-binding specificity and only orchestrates the expression of ACR2 and ACR3 genes. In the DNA-binding basic region, Yap8 has three distinct amino acids residues, Leu26, Ser29 and Asn31, at sites of highly conserved positions in the other Yap family of transcriptional regulators and Pap1 of Schizosaccharomyces pombe. To evaluate whether these residues are relevant to Yap8 specificity, we first built a homology model of the complex Yap8bZIP-DNA based on Pap1-DNA crystal structure. Several Yap8 mutants were then generated in order to confirm the contribution of the residues predicted to interact with DNA. Using bioinformatics analysis together with in vivo and in vitro approaches, we have identified several conserved residues critical for Yap8-DNA binding. Moreover, our data suggest that Leu26 is required for Yap8 binding to DNA and that this residue together with Asn31, hinder Yap1 response element recognition by Yap8, thus narrowing its DNA-binding specificity. Furthermore our results point to a role of these two amino acids in the stability of the Yap8-DNA complex.

Contribution of TyrB26 to the Function and Stability of Insulin

PubMed Central

Pandyarajan, Vijay; Phillips, Nelson B.; Rege, Nischay; Lawrence, Michael C.; Whittaker, Jonathan; Weiss, Michael A.

2016-01-01

Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of TyrB26, a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, non-aromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakest-binding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the GluB26 analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although TyrB26 is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences. PMID:27129279

Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

PubMed

Zhang, Limeng; Zhou, Xue; Fan, Ziyao; Tang, Wei; Chen, Liang; Dai, Jian; Wei, Yuhua; Zhang, Jianxin; Yang, Xuan; Yang, Xijing; Liu, Daolong; Yu, Liquan; Zhang, Hua; Wu, Zhijun; Yu, Yongzhong; Sun, Hunan; Cui, Yudong

2015-01-01

Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phosphotransfer reactions of the CbbRRS three-protein two- component system from Rhodopseudomonas palustris CGA010 appear to be controlled by an internal molecular switch on the sensor kinase.

PubMed

Romagnoli, Simona; Tabita, F Robert

2007-01-01

The CbbRRS system is an atypical three-protein two-component system that modulates the expression of the cbb(I) CO(2) fixation operon of Rhodopseudomonas palustris, possibly in response to a redox signal. It consists of a membrane-bound hybrid sensor kinase, CbbSR, with a transmitter and receiver domain, and two response regulator proteins, CbbRR1 and CbbRR2. No detectable helix-turn-helix DNA binding domain is associated with either response regulator, but an HPt domain and a second receiver domain are predicted at the C-terminal region of CbbRR1 and CbbRR2, respectively. The abundance of conserved residues predicted to participate in a His-Asp phosphorelay raised the question of their de facto involvement. In this study, the role of the multiple receiver domains was elucidated in vitro by generating site-directed mutants of the putative conserved residues. Distinct phosphorylation patterns were obtained with two truncated versions of the hybrid sensor kinase, CbbSR(T189) and CbbSR(R96) (CbbSR beginning at residues T189 and R96, respectively). These constructs also exhibited substantially different affinities for ATP and phosphorylation stability, which was found to be dependent on a conserved Asp residue (Asp-696) within the kinase receiver domain. Asp-696 also played an important role in defining the specificity of phosphorylation for response regulators CbbRR1 or CbbRR2, and this residue appeared to act in conjunction with residues within the region from Arg-96 to Thr-189 at the N terminus of the sensor kinase. The net effect of concerted interactions at these distinct regions of CbbSR created an internal molecular switch that appears to coordinate a unique branched phosphorelay system.

Influence of hydrophobic and electrostatic residues on SARS-coronavirus S2 protein stability: Insights into mechanisms of general viral fusion and inhibitor design

PubMed Central

Aydin, Halil; Al-Khooly, Dina; Lee, Jeffrey E

2014-01-01

Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS-coronavirus (SARS-CoV). SARS-CoV entry is facilitated by the spike protein (S), which consists of an N-terminal domain (S1) responsible for cellular attachment and a C-terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS-CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site-directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH-independent retroviral fusion proteins, SARS-CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS-CoV S2 analysis showed that specific hydrophobic positions at the C-terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C-terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C-terminal hydrophobic residues led us to identify a 42-residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation = 0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections. PMID:24519901

Influence of hydrophobic and electrostatic residues on SARS-coronavirus S2 protein stability: insights into mechanisms of general viral fusion and inhibitor design.

PubMed

Aydin, Halil; Al-Khooly, Dina; Lee, Jeffrey E

2014-05-01

Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS-coronavirus (SARS-CoV). SARS-CoV entry is facilitated by the spike protein (S), which consists of an N-terminal domain (S1) responsible for cellular attachment and a C-terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS-CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site-directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH-independent retroviral fusion proteins, SARS-CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS-CoV S2 analysis showed that specific hydrophobic positions at the C-terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C-terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C-terminal hydrophobic residues led us to identify a 42-residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation=0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections. © 2014 The Protein Society.

Two different groups of signal sequence in M-superfamily conotoxins.

PubMed

Wang, Qi; Jiang, Hui; Han, Yu-Hong; Yuan, Duo-Duo; Chi, Cheng-Wu

2008-04-01

M-superfamily conotoxins can be divided into four branches (M-1, M-2, M-3 and M-4) according to the number of amino acid residues in the third Cys loop. In general, it is widely accepted that the conotoxin signal peptides of each superfamily are strictly conserved. Recently, we cloned six cDNAs of novel M-superfamily conotoxins from Conus leopardus, Conus marmoreus and Conus quercinus, belonging to either M-1 or M-3 branch. These conotoxins, judging from the putative peptide sequences deducted from cDNAs, are rich in acidic residues and share highly conserved signal and pro-peptide region. However, they are quite different from the reported conotoxins of M-2 and M-4 branches even in their signal peptides, which in general are considered highly conserved for each superfamily of conotoxins. The signal sequences of M-1 and M-3 conotoxins composed of 24 residues start with MLKMGVVL-, while those of M-2 and M-4 conotoxins composed of 25 residues start with MMSKLGVL-. It is another example that different types of signal peptides can exist within a superfamily besides the I-conotoxin superfamily. In addition to the different disulfide connectivity of M-1 conotoxins from that of M-4 or M-2 conotoxins, the sequence alignment, preferential Cys codon usage and phylogenetic tree analysis suggest that M-1 and M-3 conotoxins have much closer relationship, being different from the conotoxins of other two branches (M-4 and M-2) of M-superfamily.

Effect of roller/crimper designs in terminating rye cover crop in small-scale conservation systems

USDA-ARS?s Scientific Manuscript database

In recent years, use of cover crops in no-till organic production systems has steadily increased. When cover crops are terminated at an appropriate growth stage, the unincorporated residue mulch protects the soil from erosion, runoff, soil compaction, and weed pressure, and conserves soil water. In ...

Soil nitrogen dynamics and leaching under conservation tillage in the Atlantic Coastal Plain, Georgia, USA

USDA-ARS?s Scientific Manuscript database

Conservation tillage (CsT) involves management that reduces soil erosion by maintaining crop residue cover on farm fields. Typically, both infiltration and soil organic matter increase over time with CsT practices. We compared the impact of a commonly used CsT practice, strip tillage (ST), to conven...

Effects of integrated polyethylene and cover crop mulch, conservation tillage, and herbicide application on weed control, yield, and economic returns in watermelon

USDA-ARS?s Scientific Manuscript database

The lack of conservation adoption in watermelon production reflects the lack of field practice recommendations addressing the challenges associated with producing vegetables in minimally disturbed cover crop residues. A three year watermelon experiment was established in fall 2013 to evaluate integr...

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation*

PubMed Central

Zattas, Dimitrios; Berk, Jason M.; Kreft, Stefan G.; Hochstrasser, Mark

2016-01-01

Specific proteins are modified by ubiquitin at the endoplasmic reticulum (ER) and are degraded by the proteasome, a process referred to as ER-associated protein degradation. In Saccharomyces cerevisiae, two principal ER-associated protein degradation ubiquitin ligases (E3s) reside in the ER membrane, Doa10 and Hrd1. The membrane-embedded Doa10 functions in the degradation of substrates in the ER membrane, nuclear envelope, cytoplasm, and nucleoplasm. How most E3 ligases, including Doa10, recognize their protein substrates remains poorly understood. Here we describe a previously unappreciated but highly conserved C-terminal element (CTE) in Doa10; this cytosolically disposed 16-residue motif follows the final transmembrane helix. A conserved CTE asparagine residue is required for ubiquitylation and degradation of a subset of Doa10 substrates. Such selectivity suggests that the Doa10 CTE is involved in substrate discrimination and not general ligase function. Functional conservation of the CTE was investigated in the human ortholog of Doa10, MARCH6 (TEB4), by analyzing MARCH6 autoregulation of its own degradation. Mutation of the conserved Asn residue (N890A) in the MARCH6 CTE stabilized the normally short lived enzyme to the same degree as a catalytically inactivating mutation (C9A). We also report the localization of endogenous MARCH6 to the ER using epitope tagging of the genomic MARCH6 locus by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. These localization and CTE analyses support the inference that MARCH6 and Doa10 are functionally similar. Moreover, our results with the yeast enzyme suggest that the CTE is involved in the recognition and/or ubiquitylation of specific protein substrates. PMID:27068744

Structural and functional determinants of conserved lipid interaction domains of inward rectifying Kir6.2 channels.

PubMed

Cukras, Catherine A; Jeliazkova, Iana; Nichols, Colin G

2002-06-01

All members of the inward rectifiier K(+) (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively. GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple beta-strands and a conserved alpha-helix (residues approximately 306-311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a alpha-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.

Functionally essential, invariant glutamate near the C-terminus of strand beta 5 in various (alpha/beta)8-barrel enzymes as a possible indicator of their evolutionary relatedness.

PubMed

Janecek, S; Baláz, S

1995-08-01

Twelve different (alpha/beta)8-barrel enzymes belonging to three structurally distinct families were found to contain, near the C-terminus of their strand beta 5, a conserved invariant glutamic acid residue that plays an important functional role in each of these enzymes. The search was based on the idea that a conserved sequence region of an (alpha/beta)8-barrel enzyme should be more or less conserved also in the equivalent part of the structure of the other enzymes with this folding motif owing to their mutual evolutionary relatedness. For this purpose, the sequence region around the well conserved fifth beta-strand of alpha-amylase containing catalytic glutamate (Glu230, Aspergillus oryzae alpha-amylase numbering), was used as the sequence-structural template. The isolated sequence stretches of the 12 (alpha/beta)8-barrels are discussed from both the sequence-structural and the evolutionary point of view, the invariant glutamate residue being proposed to be a joining feature of the studied group of enzymes remaining from their ancestral (alpha/beta)8-barrel.

Importance of Conserved Cysteine Residues in the Coronavirus Envelope Protein▿

PubMed Central

Lopez, Lisa A.; Riffle, Ambere J.; Pike, Steven L.; Gardner, Douglas; Hogue, Brenda G.

2008-01-01

Coronavirus envelope (E) proteins play an important, not fully understood role(s) in the virus life cycle. All E proteins have conserved cysteine residues located on the carboxy side of the long hydrophobic domain, suggesting functional significance. In this study, we confirmed that mouse hepatitis coronavirus A59 E protein is palmitoylated. To understand the role of the conserved residues and the necessity of palmitoylation, three cysteines at positions 40, 44, and 47 were changed singly and in various combinations to alanine. Double- and triple-mutant E proteins resulted in decreased virus-like particle output when coexpressed with the membrane (M) protein. Mutant E proteins were also studied in the context of a full-length infectious clone. Single-substitution viruses exhibited growth characteristics virtually identical to those of the wild-type virus, while the double-substitution mutations gave rise to viruses with less robust growth phenotypes indicated by smaller plaques and decreased virus yields. In contrast, replacement of all three cysteines resulted in crippled virus with significantly reduced yields. Triple-mutant viruses did not exhibit impairment in entry. Mutant E proteins localized properly in infected cells. A comparison of intracellular and extracellular virus yields suggested that release is only slightly impaired. E protein lacking all three cysteines exhibited an increased rate of degradation compared to that of the wild-type protein, suggesting that palmitoylation is important for the stability of the protein. Altogether, the results indicate that the conserved cysteines and presumably palmitoylation are functionally important for virus production. PMID:18184703

Conformational dynamics underlie the activity of the auxin-binding protein, Nt-abp1.

PubMed

David, K; Carnero-Diaz, E; Leblanc, N; Monestiez, M; Grosclaude, J; Perrot-Rechenmann, C

2001-09-14

The auxin-binding protein 1 (ABP1) has been proposed to be involved in the perception of the phytohormone at the plasma membrane. Site-directed mutagenesis was performed on highly conserved residues at the C terminus of ABP1 to investigate their relative importance in protein folding and activation of a functional response at the plasma membrane. Detailed analysis of the dynamic interaction of the wild-type ABP1 and mutated proteins with three distinct monoclonal antibodies recognizing conformation-dependent epitopes was performed by surface plasmon resonance. The influence of auxin on these interactions was also investigated. The Cys(177) as well as Asp(175) and Glu(176) were identified as critical residues for ABP1 folding and action at the plasma membrane. On the contrary, the C-terminal KDEL sequence was demonstrated not to be essential for auxin binding, interaction with the plasma membrane, or activation of the transduction cascade although it does appear to be involved in the stability of ABP1. Taken together, the results confirmed that ABP1 conformational change is the critical step for initiating the signal from the plasma membrane.

Identification of the srtC1 Transcription Start Site and Catalytically Essential Residues Required for Actinomyces oris T14V SrtC1 Activity

DTIC Science & Technology

2011-07-27

domain (type 2 phosphatidic acid phosphatase) and may be a PAP2 like superfamily member. In order to localize the promoter(s) for these three genes...Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 which amino acid residue(s) was critical for the enzyme activity. This enzyme possesses a...analyzed the role of eight conserved amino acid residues. The amino acids to be mutated were chosen based on the sequence alignment of several class C

Active site remodeling during the catalytic cycle in metal-dependent fructose-1,6-bisphosphate aldolases.

PubMed

Jacques, Benoit; Coinçon, Mathieu; Sygusch, Jurgen

2018-03-28

Crystal structures of two bacterial metal (Zn) dependent D-fructose 1,6-bisphosphate (FBP) aldolases in complex with substrate, analogues, and triose-P reaction products were determined to 1.5-2.0 Å resolution. The ligand complexes cryotrapped in native or mutant H. pylori aldolase crystals enabled a novel mechanistic description of FBP C 3 -C 4 bond cleavage. The reaction mechanism uses active site remodelling during the catalytic cycle implicating relocation of the Zn cofactor that is mediated by conformational changes of active site loops. Substrate binding initiates conformational changes, triggered upon P 1 -phosphate binding, which liberates the Zn chelating His180, allowing it to act as a general base for the proton abstraction at the FBP C 4 -hydroxyl group. A second zinc chelating His83 hydrogen bonds the substrate C 4 - hydroxyl group and assists cleavage by stabilizing the developing negative charge during proton abstraction. Cleavage is concerted with relocation of the metal cofactor from an interior to a surface exposed site, thereby stabilizing the nascent enediolate form. Conserved residue Glu142 is essential for protonation of the enediolate form, prior to product release. A D-tagatose 1,6-bisphosphate enzymatic complex reveals how His180 mediated proton abstraction controls stereospecificity of the cleavage reaction. Recognition and discrimination of the reaction products, dihydroxyacetone-P and D-glyceraldehyde-3-P, occurs via charged hydrogen bonds between hydroxyl groups of the triose-Ps and conserved residues, Asp82 and Asp255, respectively, and are crucial aspects of the enzyme's role in gluconeogenesis. Conformational changes in mobile loops β5-α7 and β6-α8 (containing catalytic residues Glu142 and His180, respectively) drive active site remodelling enabling the relocation of the metal cofactor. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Catalysis by the second class of tRNA(m1G37) methyl transferase requires a conserved proline.

PubMed

Christian, Thomas; Evilia, Caryn; Hou, Ya-Ming

2006-06-20

The enzyme tRNA(m1G37) methyl transferase catalyzes the transfer of a methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37, which is 3' to the anticodon sequence and whose modification is important for maintaining the reading frame fidelity. While the enzyme in bacteria is highly conserved and is encoded by the trmD gene, recent studies show that the counterpart of this enzyme in archaea and eukarya, encoded by the trm5 gene, is unrelated to trmD both in sequence and in structure. To further test this prediction, we seek to identify residues in the second class of tRNA(m1G37) methyl transferase that are required for catalysis. Such residues should provide mechanistic insights into the distinct structural origins of the two classes. Using the Trm5 enzyme of the archaeon Methanocaldococcus jannaschii (previously MJ0883) as an example, we have created mutants to test many conserved residues for their catalytic potential and substrate-binding capabilities with respect to both AdoMet and tRNA. We identified that the proline at position 267 (P267) is a critical residue for catalysis, because substitution of this residue severely decreases the kcat of the methylation reaction in steady-state kinetic analysis, and the k(chem) in single turnover kinetic analysis. However, substitution of P267 has milder effect on the Km and little effect on the Kd of either substrate. Because P267 has no functional side chain that can directly participate in the chemistry of methyl transfer, we suggest that its role in catalysis is to stabilize conformations of enzyme and substrates for proper alignment of reactive groups at the enzyme active site. Sequence analysis shows that P267 is embedded in a peptide motif that is conserved among the Trm5 family, but absent from the TrmD family, supporting the notion that the two families are descendants of unrelated protein structures.

Discovery and structure determination of the orphan enzyme isoxanthopterin deaminase .

PubMed

Hall, Richard S; Agarwal, Rakhi; Hitchcock, Daniel; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Raushel, Frank M

2010-05-25

Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a ( gi|44585104 ) and NYSGXRC-9236b ( gi|44611670 ), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 A resolution (Protein Data Bank entry 2PAJ ). This protein folds as a distorted (beta/alpha)(8) barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s(-1), 8.0 muM, and 1.3 x 10(5) M(-1) s(-1) (k(cat), K(m), and k(cat)/K(m), respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9 ). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes which are predicted to deaminate isoxanthopterin.

Tolerance of the archaeal Sac7d scaffold protein to alternative library designs: characterization of anti-immunoglobulin G Affitins.

PubMed

Béhar, Ghislaine; Bellinzoni, Marco; Maillasson, Mike; Paillard-Laurance, Lauranne; Alzari, Pedro M; He, Xuemei; Mouratou, Barbara; Pecorari, Frédéric

2013-04-01

Engineered protein scaffolds have received considerable attention as alternatives to antibodies in both basic and applied research, as they can offer superior biophysical properties often associated with a simpler molecular organization. Sac7d has been demonstrated as an effective scaffold for molecular recognition. Here, we used the initial L1 'flat surface' library constructed by randomization of 14 residues, to identify ligands specific for human immunoglobulin G. To challenge the plasticity of the Sac7d protein scaffold, we designed the alternative L2 'flat surface & loops' library whereof only 10 residues are randomized. Representative binders (Affitins) of the two libraries exhibited affinities in the low nanomolar range and were able to recognize different epitopes within human immunoglobulin G. These Affitins were stable up to pH 12 while largely conserving other favorable properties of Sac7d protein, such as high expression yields in Escherichia coli, solubility, thermal stability up to 80.7°C, and acidic stability (pH 0). In agreement with our library designs, mutagenesis study revealed two distinct binding areas, one including loops. Together, our results indicate that the Sac7d scaffold tolerates alternative library designs, which further expands the diversity of Affitins and may provide a general way to create tailored affinity tools for demanding applications.

A Conserved Asparagine in a P-type Proton Pump Is Required for Efficient Gating of Protons*

PubMed Central

Ekberg, Kira; Wielandt, Alex G.; Buch-Pedersen, Morten J.; Palmgren, Michael G.

2013-01-01

The minimal proton pumping machinery of the Arabidopsis thaliana P-type plasma membrane H+-ATPase isoform 2 (AHA2) consists of an aspartate residue serving as key proton donor/acceptor (Asp-684) and an arginine residue controlling the pKa of the aspartate. However, other important aspects of the proton transport mechanism such as gating, and the ability to occlude protons, are still unclear. An asparagine residue (Asn-106) in transmembrane segment 2 of AHA2 is conserved in all P-type plasma membrane H+-ATPases. In the crystal structure of the plant plasma membrane H+-ATPase, this residue is located in the putative ligand entrance pathway, in close proximity to the central proton donor/acceptor Asp-684. Substitution of Asn-106 resulted in mutant enzymes with significantly reduced ability to transport protons against a membrane potential. Sensitivity toward orthovanadate was increased when Asn-106 was substituted with an aspartate residue, but decreased in mutants with alanine, lysine, glutamine, or threonine replacement of Asn-106. The apparent proton affinity was decreased for all mutants, most likely due to a perturbation of the local environment of Asp-684. Altogether, our results demonstrate that Asn-106 is important for closure of the proton entrance pathway prior to proton translocation across the membrane. PMID:23420846

A conserved asparagine in a P-type proton pump is required for efficient gating of protons.

PubMed

Ekberg, Kira; Wielandt, Alex G; Buch-Pedersen, Morten J; Palmgren, Michael G

2013-04-05

The minimal proton pumping machinery of the Arabidopsis thaliana P-type plasma membrane H(+)-ATPase isoform 2 (AHA2) consists of an aspartate residue serving as key proton donor/acceptor (Asp-684) and an arginine residue controlling the pKa of the aspartate. However, other important aspects of the proton transport mechanism such as gating, and the ability to occlude protons, are still unclear. An asparagine residue (Asn-106) in transmembrane segment 2 of AHA2 is conserved in all P-type plasma membrane H(+)-ATPases. In the crystal structure of the plant plasma membrane H(+)-ATPase, this residue is located in the putative ligand entrance pathway, in close proximity to the central proton donor/acceptor Asp-684. Substitution of Asn-106 resulted in mutant enzymes with significantly reduced ability to transport protons against a membrane potential. Sensitivity toward orthovanadate was increased when Asn-106 was substituted with an aspartate residue, but decreased in mutants with alanine, lysine, glutamine, or threonine replacement of Asn-106. The apparent proton affinity was decreased for all mutants, most likely due to a perturbation of the local environment of Asp-684. Altogether, our results demonstrate that Asn-106 is important for closure of the proton entrance pathway prior to proton translocation across the membrane.

Conservation analysis and decomposition of residue correlation networks in the phospholipase A2 superfamily (PLA2s): Insights into the structure-function relationships of snake venom toxins.

PubMed

Oliveira, Alberto; Bleicher, Lucas; Schrago, Carlos G; Silva Junior, Floriano Paes

2018-05-01

Phospholipases A2 (PLA 2 s) comprise a superfamily of glycerophospholipids hydrolyzing enzymes present in many organisms in nature, whose catalytic activity was majorly unveiled by analysis of snake venoms. The latter have pharmaceutical and biotechnological interests and can be divided into different functional sub-classes. Our goal was to identify important residues and their relation to the functional and class-specific characteristics in the PLA 2 s family with special emphasis on snake venom PLA 2 s (svPLA 2 s). We identified such residues by conservation analysis and decomposition of residue coevolution networks (DRCN), annotated the results based on the available literature on PLA 2 s, structural analysis and molecular dynamics simulations, and related the results to the phylogenetic distribution of these proteins. A filtered alignment of PLA 2 s revealed 14 highly conserved positions and 3 sets of coevolved residues, which were annotated according to their structural or functional role. These residues are mostly involved in ligand binding and catalysis, calcium-binding, the formation of disulfide bridges and a hydrophobic cluster close to the binding site. An independent validation of the inference of structure-function relationships from our co-evolution analysis on the svPLA2s family was obtained by the analysis of the pattern of selection acting on the Viperidae and Elapidae lineages. Additionally, a molecular dynamics simulation on the Lys49 PLA 2 from Agkistrodon contortrix laticinctus was carried out to further investigate the correlation of the Lys49-Glu69 pair. Our results suggest this configuration can result in a novel conformation where the binding cavity collapses due to the approximation of two loops caused by a strong salt bridge between Glu69 and Arg34. Finally, phylogenetic analysis indicated a correlation between the presence of residues in the coevolved sets found in this analysis and the clade localization. The results provide a guide for important positions in the family of PLA 2 s, and potential new objects of investigation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Genetic and structural analyses of cytochrome P450 hydroxylases in sex hormone biosynthesis: Sequential origin and subsequent coevolution.

PubMed

Goldstone, Jared V; Sundaramoorthy, Munirathinam; Zhao, Bin; Waterman, Michael R; Stegeman, John J; Lamb, David C

2016-01-01

Biosynthesis of steroid hormones in vertebrates involves three cytochrome P450 hydroxylases, CYP11A1, CYP17A1 and CYP19A1, which catalyze sequential steps in steroidogenesis. These enzymes are conserved in the vertebrates, but their origin and existence in other chordate subphyla (Tunicata and Cephalochordata) have not been clearly established. In this study, selected protein sequences of CYP11A1, CYP17A1 and CYP19A1 were compiled and analyzed using multiple sequence alignment and phylogenetic analysis. Our analyses show that cephalochordates have sequences orthologous to vertebrate CYP11A1, CYP17A1 or CYP19A1, and that echinoderms and hemichordates possess CYP11-like but not CYP19 genes. While the cephalochordate sequences have low identity with the vertebrate sequences, reflecting evolutionary distance, the data show apparent origin of CYP11 prior to the evolution of CYP19 and possibly CYP17, thus indicating a sequential origin of these functionally related steroidogenic CYPs. Co-occurrence of the three CYPs in early chordates suggests that the three genes may have coevolved thereafter, and that functional conservation should be reflected in functionally important residues in the proteins. CYP19A1 has the largest number of conserved residues while CYP11A1 sequences are less conserved. Structural analyses of human CYP11A1, CYP17A1 and CYP19A1 show that critical substrate binding site residues are highly conserved in each enzyme family. The results emphasize that the steroidogenic pathways producing glucocorticoids and reproductive steroids are several hundred million years old and that the catalytic structural elements of the enzymes have been conserved over the same period of time. Analysis of these elements may help to identify when precursor functions linked to these enzymes first arose. Copyright © 2015 Elsevier Inc. All rights reserved.

Conversion of Conservation Tillage to Rotational Tillage to Reduce Phosphorus Losses during Snowmelt Runoff in the Canadian Prairies.

PubMed

Liu, Kui; Elliott, Jane A; Lobb, David A; Flaten, Don N; Yarotski, Jim

2014-09-01

In a preceding study, converting conventional tillage (ConvT) to conservation tillage (ConsT) was reported to decrease nitrogen (N) but to increase phosphorus (P) losses during snowmelt runoff. A field-scale study was conducted from 2004 to 2012 to determine if conversion of ConsT to rotational tillage (RotaT), where conservation tillage was interrupted by a fall tillage pass every other year, could effectively reduce P losses compared with ConsT. The RotaT study was conducted on long-term paired watersheds established in 1993. The ConvT field in the pair has remained under ConvT practice since 1993, whereas tillage was minimized on the ConsT field from 1997 until 2007. In fall 2007, RotaT was introduced to the ConsT field, and heavy-duty cultivator passes were conducted in the late fall of years 2007, 2009, and 2011. Runoff volume and nutrient content were monitored at the edge of the two fields, and soil and crop residue samples were taken in each field. Greater soil Olsen P and more P released from crop residue are likely the reasons for the increased P losses in the ConsT treatment (2004-2007) relative to the ConvT treatment (2004-2007). Analysis of covariance indicated that, compared with ConsT (2004-2007), RotaT (2008-2012) increased the concentrations of dissolved organic carbon (DOC) by 62%, total dissolved N (TDN) by 190%, and total N (TN) by 272% and increased the loads of DOC by 34%, TDN by 34%, and TN by 60%. However, RotaT (2008-2012) decreased soil test P in surface soil, P released from crop residue, and duration of runoff compared with ConsT (2004-2007) and thus decreased the concentrations of total dissolved P (TDP) by 46% and total P (TP) by 38% and decreased the loads of TDP by 56% and TP by 42%. In the Canadian Prairies, where P is a major environmental concern compared with N, RotaT was demonstrated to be an effective practice to reduce P losses compared with ConsT. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Study on Plastic Deformation Characteristics of Shot Peening of Ni-Based Superalloy GH4079

NASA Astrophysics Data System (ADS)

Zhong, L. Q.; Liang, Y. L.; Hu, H.

2017-09-01

In this paper, the X-ray stress diffractometer, surface roughness tester, field emission scanning electron microscope(SEM), dynamic ultra-small microhardness tester were used to measure the surface residual stress and roughness, topography and surface hardness changes of GH4079 superalloy, which was processed by metallographic grinding, turning, metallographic grinding +shot peening and turning + shot peening. Analysized the effects of shot peening parameters on shot peening plastic deformation features; and the effects of the surface state before shot peening on shot peening plastic deformation characteristics. Results show that: the surface residual compressive stress, surface roughness and surface hardness of GH4079 superalloy were increased by shot peening, in addition, the increment of the surface residual compressive stress, surface roughness and surface hardness induced by shot peening increased with increasing shot peening intensity, shot peening time, shot peening pressure and shot hardness, but harden layer depth was not affected considerably. The more plastic deformation degree of before shot peening surface state, the less increment of the surface residual compressive stress, surface roughness and surface hardness induced by shot peening.

Standoff spectroscopy using a conditioned target

DOEpatents

Van Neste, Charles W [Kingston, TN; Morales-Rodriguez, Marissa E [Knoxville, TN; Senesac, Lawrence R [Knoxville, TN; Thundat, Thomas G [Knoxville, TN

2011-12-20

A system and method are disclosed for standoff spectroscopy of molecules (e.g. from a residue) on a surface from a distance. A source emits radiation that modifies or conditions the residue, such as through photodecomposition. A spectral generating source measures a spectrum of the residue before and after the residue is exposed to the radiation from that source. The two spectra are compared to produce a distinct identification of the residues on the surface or identify certain properties of the residue.

Coevolution at protein complex interfaces can be detected by the complementarity trace with important impact for predictive docking

PubMed Central

Madaoui, Hocine; Guerois, Raphaël

2008-01-01

Protein surfaces are under significant selection pressure to maintain interactions with their partners throughout evolution. Capturing how selection pressure acts at the interfaces of protein–protein complexes is a fundamental issue with high interest for the structural prediction of macromolecular assemblies. We tackled this issue under the assumption that, throughout evolution, mutations should minimally disrupt the physicochemical compatibility between specific clusters of interacting residues. This constraint drove the development of the so-called Surface COmplementarity Trace in Complex History score (SCOTCH), which was found to discriminate with high efficiency the structure of biological complexes. SCOTCH performances were assessed not only with respect to other evolution-based approaches, such as conservation and coevolution analyses, but also with respect to statistically based scoring methods. Validated on a set of 129 complexes of known structure exhibiting both permanent and transient intermolecular interactions, SCOTCH appears as a robust strategy to guide the prediction of protein–protein complex structures. Of particular interest, it also provides a basic framework to efficiently track how protein surfaces could evolve while keeping their partners in contact. PMID:18511568

Behavior of sulfur mustard in sand, concrete, and asphalt matrices: Evaporation, degradation, and decontamination.

PubMed

Jung, Hyunsook; Choi, Seungki

2017-10-15

The evaporation, degradation, and decontamination of sulfur mustard on environmental matrices including sand, concrete, and asphalt are described. A specially designed wind tunnel and thermal desorber in combination with gas chromatograph (GC) produced profiles of vapor concentration obtained from samples of the chemical agent deposited as a drop on the surfaces of the matrices. The matrices were exposed to the chemical agent at room temperature, and the degradation reactions were monitored and characterized. A vapor emission test was also performed after a decontamination process. The results showed that on sand, the drop of agent spread laterally while evaporating. On concrete, the drop of the agent was absorbed immediately into the matrix while spreading and evaporating. However, the asphalt surface conserved the agent and slowly released parts of the agent over an extended period of time. The degradation reactions of the agent followed pseudo first order behavior on the matrices. Trace amounts of the residual agent present at the surface were also released as vapor after decontamination, posing a threat to the exposed individual and environment.

Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II.

PubMed

Criscitiello, Michael F; Ohta, Yuko; Graham, Matthew D; Eubanks, Jeannine O; Chen, Patricia L; Flajnik, Martin F

2012-03-01

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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...

Crystal structure of the Tum1 protein from the yeast Saccharomyces cerevisiae.

PubMed

Qiu, Rui; Wang, Fengbin; Liu, Meiruo; Lou, Tiantian; Ji, Chaoneng

2012-11-01

Yeast tRNA-thiouridine modification protein 1 (Tum1) plays essential role in the sulfur transfer process of Urm1 system, which in turn is involved in many important cellular processes. In the rhodanese-like domain (RLD), conserved cysteine residue is proved to be the centre of active site of sulfurtransferases and crucial for the substrate recognition. In this report, we describe the crystal structure of Tum1 protein at 1.90 A resolution which, despite consisting of two RLDs, has only one conserved cysteine residue in the C-terminal RLD. An unaccounted electron density is found near the active site, which might point to the new cofactor in the sulfur transfer mechanism.

Finite Element Simulation of Shot Peening: Prediction of Residual Stresses and Surface Roughness

NASA Astrophysics Data System (ADS)

Gariépy, Alexandre; Perron, Claude; Bocher, Philippe; Lévesque, Martin

Shot peening is a surface treatment that consists of bombarding a ductile surface with numerous small and hard particles. Each impact creates localized plastic strains that permanently stretch the surface. Since the underlying material constrains this stretching, compressive residual stresses are generated near the surface. This process is commonly used in the automotive and aerospace industries to improve fatigue life. Finite element analyses can be used to predict residual stress profiles and surface roughness created by shot peening. This study investigates further the parameters and capabilities of a random impact model by evaluating the representative volume element and the calculated stress distribution. Using an isotropic-kinematic hardening constitutive law to describe the behaviour of AA2024-T351 aluminium alloy, promising results were achieved in terms of residual stresses.

Residual stress in glass: indentation crack and fractography approaches.

PubMed

Anunmana, Chuchai; Anusavice, Kenneth J; Mecholsky, John J

2009-11-01

To test the hypothesis that the indentation crack technique can determine surface residual stresses that are not statistically significantly different from those determined from the analytical procedure using surface cracks, the four-point flexure test, and fracture surface analysis. Soda-lime-silica glass bar specimens (4 mm x 2.3 mm x 28 mm) were prepared and annealed at 650 degrees C for 30 min before testing. The fracture toughness values of the glass bars were determined from 12 specimens based on induced surface cracks, four-point flexure, and fractographic analysis. To determine the residual stress from the indentation technique, 18 specimens were indented under 19.6N load using a Vickers microhardness indenter. Crack lengths were measured within 1 min and 24h after indentation, and the measured crack lengths were compared with the mean crack lengths of annealed specimens. Residual stress was calculated from an equation developed for the indentation technique. All specimens were fractured in a four-point flexure fixture and the residual stress was calculated from the strength and measured crack sizes on the fracture surfaces. The results show that there was no significant difference between the residual stresses calculated from the two techniques. However, the differences in mean residual stresses calculated within 1 min compared with those calculated after 24h were statistically significant (p=0.003). This study compared the indentation technique with the fractographic analysis method for determining the residual stress in the surface of soda-lime-silica glass. The indentation method may be useful for estimating residual stress in glass.

Residual stress in glass: indentation crack and fractography approaches

PubMed Central

Anunmana, Chuchai; Anusavice, Kenneth J.; Mecholsky, John J.

2009-01-01

Objective To test the hypothesis that the indentation crack technique can determine surface residual stresses that are not statistically significantly different from those determined from the analytical procedure using surface cracks, the four-point flexure test, and fracture surface analysis. Methods Soda-lime-silica glass bar specimens (4 mm × 2.3 mm × 28 mm) were prepared and annealed at 650 °C for 30 min before testing. The fracture toughness values of the glass bars were determined from 12 specimens based on induced surface cracks, four-point flexure, and fractographic analysis. To determine the residual stress from the indentation technique, 18 specimens were indented under 19.6 N load using a Vickers microhardness indenter. Crack lengths were measured within 1 min and 24 h after indentation, and the measured crack lengths were compared with the mean crack lengths of annealed specimens. Residual stress was calculated from an equation developed for the indentation technique. All specimens were fractured in a four-point flexure fixture and the residual stress was calculated from the strength and measured crack sizes on the fracture surfaces. Results The results show that there was no significant difference between the residual stresses calculated from the two techniques. However, the differences in mean residual stresses calculated within 1 min compared with those calculated after 24 h were statistically significant (p=0.003). Significance This study compared the indentation technique with the fractographic analysis method for determining the residual stress in the surface of soda-lime silica glass. The indentation method may be useful for estimating residual stress in glass. PMID:19671475

Molecular docking of superantigens with class II major histocompatibility complex proteins.

PubMed

Olson, M A; Cuff, L

1997-01-01

The molecular recognition of two superantigens with class II major histocompatibility complex molecules was simulated by using protein-protein docking. Superantigens studied were staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1) in their crystallographic assemblies with HLA-DR1. Rigid-body docking was performed sampling configurational space of the interfacial surfaces by employing a strategy of partitioning the contact regions on HLA-DR1 into separate molecular recognition units. Scoring of docked conformations was based on an electrostatic continuum model evaluated with the finite-difference Poisson-Boltzmann method. Estimates of nonpolar contributions were derived from the buried molecular surface areas. We found for both superantigens that docking the HLA-DR1 surface complementary with the SEB and TSST-1 contact regions containing a homologous hydrophobic surface loop provided sufficient recognition for the reconstitution of native-like conformers exhibiting the highest-scoring free energies. For the SEB complex, the calculations were successful in reproducing the total association free energy. A comparison of the free-energy determinants of the conserved hydrophobic contact residue indicates functional similarity between the two proteins for this interface. Though both superantigens share a common global association mode, differences in binding topology distinguish the conformational specificities underlying recognition.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk

The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of humanmore » Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.« less

A coupled creep plasticity model for residual stress relaxation of a shot-peened nickel-based superalloy

NASA Astrophysics Data System (ADS)

Buchanan, Dennis J.; John, Reji; Brockman, Robert A.; Rosenberger, Andrew H.

2010-01-01

Shot peening is a commonly used surface treatment process that imparts compressive residual stresses into the surface of metal components. Compressive residual stresses retard initiation and growth of fatigue cracks. During component loading history, shot-peened residual stresses may change due to thermal exposure, creep, and cyclic loading. In these instances, taking full credit for compressive residual stresses would result in a nonconservative life prediction. This article describes a methodical approach for characterizing and modeling residual stress relaxation under elevated temperature loading, near and above the monotonic yield strength of INI 00. The model incorporates the dominant creep deformation mechanism, coupling between the creep and plasticity models, and effects of prior plastic strain to simulate surface treatment deformation.

On the residual stress modeling of shot-peened AISI 4340 steel: finite element and response surface methods

NASA Astrophysics Data System (ADS)

Asgari, Ali; Dehestani, Pouya; Poruraminaie, Iman

2018-02-01

Shot peening is a well-known process in applying the residual stress on the surface of industrial parts. The induced residual stress improves fatigue life. In this study, the effects of shot peening parameters such as shot diameter, shot speed, friction coefficient, and the number of impacts on the applied residual stress will be evaluated. To assess these parameters effect, firstly the shot peening process has been simulated by finite element method. Then, effects of the process parameters on the residual stress have been evaluated by response surface method as a statistical approach. Finally, a strong model is presented to predict the maximum residual stress induced by shot peening process in AISI 4340 steel. Also, the optimum parameters for the maximum residual stress are achieved. The results indicate that effect of shot diameter on the induced residual stress is increased by increasing the shot speed. Also, enhancing the friction coefficient magnitude always cannot lead to increase in the residual stress.

Triaxial Measurement Method for Analysis of Residual Stress after High Feed Milling by X-Ray Diffraction

NASA Astrophysics Data System (ADS)

Čuma, Matúš; Török, Jozef; Telišková, Monika

2016-12-01

Surface integrity is a broad term which includes various quality factors affecting the functional properties of parts. Residual stress is one of these factors. Machining generates residual stresses in the surface and subsurface layers of the structural elements. X-ray diffractometry is a non-destructive method applicable for the measurement of residual stresses in surface and subsurface layers of components. The article is focused on the non-destructive progressive method of triaxial measurement of residual stress after machining the surface of sample by high feed milling technology. Significance of triaxial measuring is the capability of measuring in different angles so it is possible to acquire stress tensor containing normal and shear stress components acting in the spot of measuring, using a Cartesian coordinate system.

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

PubMed Central

de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A.; Enghild, Jan J.; Thøgersen, Ida B.; Gao, Jinlong; Kwan, Ann H.; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F. Xavier; Nguyen, Ky-Anh; Potempa, Jan

2016-01-01

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes.

PubMed

Nandy, Suman Kumar; Seal, Alpana

2016-01-01

Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases.

Identification of Group B Streptococcal Sip Protein, Which Elicits Cross-Protective Immunity

PubMed Central

Brodeur, Bernard R.; Boyer, Martine; Charlebois, Isabelle; Hamel, Josée; Couture, France; Rioux, Clément R.; Martin, Denis

2000-01-01

A protein of group B streptococci (GBS), named Sip for surface immunogenic protein, which is distinct from previously described surface proteins, was identified after immunological screening of a genomic library. Immunoblots using a Sip-specific monoclonal antibody indicated that a protein band with an approximate molecular mass of 53 kDa which did not vary in size was present in every GBS strain tested. Representatives of all nine GBS serotypes were included in the panel of strains. Cloning and sequencing of the sip gene revealed an open reading frame of 1,305 nucleotides coding for a polypeptide of 434 amino acid residues, with a calculated pI of 6.84 and molecular mass of 45.5 kDa. Comparison of the nucleotide sequences from six different strains confirmed with 98% identity that the sip gene is highly conserved among GBS isolates. N-terminal amino acid sequencing also indicated the presence of a 25-amino-acid signal peptide which is cleaved in the mature protein. More importantly, immunization with the recombinant Sip protein efficiently protected CD-1 mice against deadly challenges with six GBS strains of serotypes Ia/c, Ib, II/R, III, V, and VI. The data presented in this study suggest that this highly conserved protein induces cross-protective immunity against GBS infections and emphasize its potential as a universal vaccine candidate. PMID:10992461

A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL Apical Domain with implications for substrate interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Q. X. H.; Dementieva, I. S. D.; Walsh, M. A. W.

2001-02-23

A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (T{sub m}>100{sup o}C as evaluated by circular dichroism) is at least 35{sup o}C greater than that of the E. coli apical domain (T{sub m}=65{sup o}C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 {angstrom} using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 {angstrom}more » based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the 'Anfinsen cage' in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using {sup 15}N-{sup 1}H correlation spectra of {sup 15}N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50{sup o}C) are essentially the same and consistent with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal {alpha}-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the 'classical' peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.« less

A single amino-acid substitution in the Ets domain alters core DNA binding specificity of Ets1 to that of the related transcription factors Elf1 and E74.

PubMed

Bosselut, R; Levin, J; Adjadj, E; Ghysdael, J

1993-11-11

Ets proteins form a family of sequence specific DNA binding proteins which bind DNA through a 85 aminoacids conserved domain, the Ets domain, whose sequence is unrelated to any other characterized DNA binding domain. Unlike all other known Ets proteins, which bind specific DNA sequences centered over either GGAA or GGAT core motifs, E74 and Elf1 selectively bind to GGAA corecontaining sites. Elf1 and E74 differ from other Ets proteins in three residues located in an otherwise highly conserved region of the Ets domain, referred to as conserved region III (CRIII). We show that a restricted selectivity for GGAA core-containing sites could be conferred to Ets1 upon changing a single lysine residue within CRIII to the threonine found in Elf1 and E74 at this position. Conversely, the reciprocal mutation in Elf1 confers to this protein the ability to bind to GGAT core containing EBS. This, together with the fact that mutation of two invariant arginine residues in CRIII abolishes DNA binding, indicates that CRIII plays a key role in Ets domain recognition of the GGAA/T core motif and lead us to discuss a model of Ets proteins--core motif interaction.

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

PubMed Central

de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

2015-01-01

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. PMID:26481363

The juxtamembrane regions of human receptor tyrosine kinases exhibit conserved interaction sites with anionic lipids

PubMed Central

Hedger, George; Sansom, Mark S. P.; Koldsø, Heidi

2015-01-01

Receptor tyrosine kinases (RTKs) play a critical role in diverse cellular processes and their activity is regulated by lipids in the surrounding membrane, including PIP2 (phosphatidylinositol-4,5-bisphosphate) in the inner leaflet, and GM3 (monosialodihexosylganglioside) in the outer leaflet. However, the precise details of the interactions at the molecular level remain to be fully characterised. Using a multiscale molecular dynamics simulation approach, we comprehensively characterise anionic lipid interactions with all 58 known human RTKs. Our results demonstrate that the juxtamembrane (JM) regions of RTKs are critical for inducing clustering of anionic lipids, including PIP2, both in simple asymmetric bilayers, and in more complex mixed membranes. Clustering is predominantly driven by interactions between a conserved cluster of basic residues within the first five positions of the JM region, and negatively charged lipid headgroups. This highlights a conserved interaction pattern shared across the human RTK family. In particular predominantly the N-terminal residues of the JM region are involved in the interactions with PIP2, whilst residues within the distal JM region exhibit comparatively less lipid specificity. Our results suggest that JM–lipid interactions play a key role in RTK structure and function, and more generally in the nanoscale organisation of receptor-containing cell membranes. PMID:25779975

Manure and residue inputs maintained SOC in conservation production systems in the Upper Midwest

USDA-ARS?s Scientific Manuscript database

Conservation production systems are needed in the upper Midwest to slow down soil and nutrient loss through tillage-induced erosion. However, due to the cool, wet climate, producers are reluctant to adapt no-till strategies. With focus on strip-tillage (ST) and a diverse four-year crop rotation (4y...

A gain-of-function mutation in the M-domain of cardiac myosin-binding protein-C increases binding to actin.

PubMed

Bezold, Kristina L; Shaffer, Justin F; Khosa, Jaskiran K; Hoye, Elaine R; Harris, Samantha P

2013-07-26

The M-domain is the major regulatory subunit of cardiac myosin-binding protein-C (cMyBP-C) that modulates actin and myosin interactions to influence muscle contraction. However, the precise mechanism(s) and the specific residues involved in mediating the functional effects of the M-domain are not fully understood. Positively charged residues adjacent to phosphorylation sites in the M-domain are thought to be critical for effects of cMyBP-C on cross-bridge interactions by mediating electrostatic binding with myosin S2 and/or actin. However, recent structural studies revealed that highly conserved sequences downstream of the phosphorylation sites form a compact tri-helix bundle. Here we used site-directed mutagenesis to probe the functional significance of charged residues adjacent to the phosphorylation sites and conserved residues within the tri-helix bundle. Results confirm that charged residues adjacent to phosphorylation sites and residues within the tri-helix bundle are important for mediating effects of the M-domain on contraction. In addition, four missense variants within the tri-helix bundle that are associated with human hypertrophic cardiomyopathy caused either loss-of-function or gain-of-function effects on force. Importantly, the effects of the gain-of-function variant, L348P, increased the affinity of the M-domain for actin. Together, results demonstrate that functional effects of the M-domain are not due solely to interactions with charged residues near phosphorylatable serines and provide the first demonstration that the tri-helix bundle contributes to the functional effects of the M-domain, most likely by binding to actin.

DRoP: a water analysis program identifies Ras-GTP-specific pathway of communication between membrane-interacting regions and the active site.

PubMed

Kearney, Bradley M; Johnson, Christian W; Roberts, Daniel M; Swartz, Paul; Mattos, Carla

2014-02-06

Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis

PubMed Central

Lohman, Danielle C.; Forouhar, Farhad; Beebe, Emily T.; Stefely, Matthew S.; Minogue, Catherine E.; Ulbrich, Arne; Stefely, Jonathan A.; Sukumar, Shravan; Luna-Sánchez, Marta; Jochem, Adam; Lew, Scott; Seetharaman, Jayaraman; Xiao, Rong; Wang, Huang; Westphall, Michael S.; Wrobel, Russell L.; Everett, John K.; Mitchell, Julie C.; López, Luis C.; Coon, Joshua J.; Tong, Liang; Pagliarini, David J.

2014-01-01

Coenzyme Q (CoQ) is an isoprenylated quinone that is essential for cellular respiration and is synthesized in mitochondria by the combined action of at least nine proteins (COQ1–9). Although most COQ proteins are known to catalyze modifications to CoQ precursors, the biochemical role of COQ9 remains unclear. Here, we report that a disease-related COQ9 mutation leads to extensive disruption of the CoQ protein biosynthetic complex in a mouse model, and that COQ9 specifically interacts with COQ7 through a series of conserved residues. Toward understanding how COQ9 can perform these functions, we solved the crystal structure of Homo sapiens COQ9 at 2.4 Å. Unexpectedly, our structure reveals that COQ9 has structural homology to the TFR family of bacterial transcriptional regulators, but that it adopts an atypical TFR dimer orientation and is not predicted to bind DNA. Our structure also reveals a lipid-binding site, and mass spectrometry-based analyses of purified COQ9 demonstrate that it associates with multiple lipid species, including CoQ itself. The conserved COQ9 residues necessary for its interaction with COQ7 comprise a surface patch around the lipid-binding site, suggesting that COQ9 might serve to present its bound lipid to COQ7. Collectively, our data define COQ9 as the first, to our knowledge, mammalian TFR structural homolog and suggest that its lipid-binding capacity and association with COQ7 are key features for enabling CoQ biosynthesis. PMID:25339443

The Structure of an Unconventional HD-GYP Protein from Bdellovibrio Reveals the Roles of Conserved Residues in this Class of Cyclic-di-GMP Phosphodiesterases

PubMed Central

Lovering, Andrew L.; Capeness, Michael J.; Lambert, Carey; Hobley, Laura; Sockett, R. Elizabeth

2011-01-01

ABSTRACT Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins. PMID:21990613

Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jinsil; Ha, Hye-Jeong; Kim, Sujin

Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that anmore » rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.« less

Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.

PubMed

Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

2010-10-01

Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

Effectiveness of Commercial and Homemade Washing Agents in Removing Pesticide Residues on and in Apples.

PubMed

Yang, Tianxi; Doherty, Jeffery; Zhao, Bin; Kinchla, Amanda J; Clark, John M; He, Lili

2017-11-08

Removal of pesticide residues from fresh produce is important to reduce pesticide exposure to humans. This study investigated the effectiveness of commercial and homemade washing agents in the removal of surface and internalized pesticide residues from apples. Surface-enhanced Raman scattering (SERS) mapping and liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were used to determine the effectiveness of different washing agents in removing pesticide residues. Surface pesticide residues were most effectively removed by sodium bicarbonate (baking soda, NaHCO 3 ) solution when compared to either tap water or Clorox bleach. Using a 10 mg/mL NaHCO 3 washing solution, it took 12 and 15 min to completely remove thiabendazole or phosmet surface residues, respectively, following a 24 h exposure to these pesticides, which were applied at a concentration of 125 ng/cm 2 . LC-MS/MS results showed, however, that 20% of applied thiabendazole and 4.4% of applied phosmet had penetrated into the apples following the 24 h exposure. Thiabendazole, a systemic pesticide, penetrated 4-fold deeper into the apple peel than did phosmet, a non-systemic pesticide, which led to more thiabendazole residues inside the apples, which could not be washed away using the NaHCO 3 washing solution. This study gives us the information that the standard postharvest washing method using Clorox bleach solution for 2 min is not an effective means to completely remove pesticide residues on the surface of apples. The NaHCO 3 method is more effective in removing surface pesticide residues on apples. In the presence of NaHCO 3 , thiabendazole and phosmet can degrade, which assists the physical removal force of washing. However, the NaHCO 3 method was not completely effective in removing residues that have penetrated into the apple peel. The overall effectiveness of the method to remove all pesticide residues diminished as pesticides penetrated deeper into the fruit. In practical application, washing apples with NaHCO 3 solution can reduce pesticides mostly from the surface. Peeling is more effective to remove the penetrated pesticides; however, bioactive compounds in the peels will become lost too.

γδ T cell receptors recognize the non-classical major histocompatibility complex (MHC) molecule T22 via conserved anchor residues in a MHC peptide-like fashion.

PubMed

Sandstrom, Andrew; Scharf, Louise; McRae, Gabrielle; Hawk, Andrew J; Meredith, Stephen C; Adams, Erin J

2012-02-17

The molecular mechanisms by which γδ T cells recognize ligand remain a mystery. The non-classical MHC molecule T22 represents the best characterized ligand for murine γδ T cells, with a motif (W … EGYEL) present in the γδ T cell receptor complementary-determining region 3δ (CDR3δ) loop mediating γδ T cell recognition of this molecule. Produced through V(D)J recombination, this loop is quite diverse, with different numbers and chemical types of amino acids between Trp and EGYEL, which have unknown functional consequences for T22 recognition. We have investigated the biophysical and structural effects of CDR3δ loop diversity, revealing a range of affinities for T22 but a common thermodynamic pattern. Mutagenesis of these CDR3δ loops defines the key anchor residues involved in T22 recognition as W … EGYEL, similar to those found for the G8 CDR3δ loop, and demonstrates that spacer residues modulate but are not required for T22 recognition. Comparison of the location of these residues in the T22 interface reveals a striking similarity to peptide anchor residues in classically presented MHC peptides, with the key Trp residue of the CDR3δ motif completing the deficient peptide-binding groove of T22. This suggests that γδ T cell recognition of T22 utilizes the conserved ligand-presenting nature of the MHC fold.

A single active site residue directs oxygenation stereospecificity in lipoxygenases: Stereocontrol is linked to the position of oxygenation

PubMed Central

Coffa, Gianguido; Brash, Alan R.

2004-01-01

Lipoxygenases are a class of dioxygenases that form hydroperoxy fatty acids with distinct positional and stereo configurations. Several amino acid residues influencing regiospecificity have been identified, whereas the basis of stereocontrol is not understood. We have now identified a single residue in the lipoxygenase catalytic domain that is important for stereocontrol; it is conserved as an Ala in S lipoxygenases and a Gly in R lipoxygenases. Our results with mutation of the conserved Ala to Gly in two S lipoxygenases (mouse 8S-LOX and human 15-LOX-2) and the corresponding Gly–Ala substitution in two R lipoxygenases (human 12R-LOX and coral 8R-LOX) reveal that the basis for R or S stereo-control also involves a switch in the position of oxygenation on the substrate. After the initial hydrogen abstraction, antarafacial oxygenation at one end or the other of the activated pair of double bonds (pentadiene) gives, for example, 8S or 12R product. The Ala residue promotes oxygenation on the reactive pentadiene at the end deep in the substrate binding pocket and S stereochemistry of the product hydroperoxide, and a Gly residue promotes oxygenation at the proximal end of the reactive pentadiene resulting in R stereochemistry. A model of lipoxygenase reaction specificity is proposed in which product regiochemistry and stereochemistry are determined by fixed relationships between substrate orientation, hydrogen abstraction, and the Gly or Ala residue we have identified. PMID:15496467

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels.

PubMed

Shang, Lijun; Tucker, Stephen J

2008-02-01

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K(+) channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the "helix-bundle crossing". However, in the inwardly rectifying (Kir) potassium channel family, the role of this "hinge" residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper "hinge" residues are in close contact with the base of the pore alpha-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the "lower" gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue.

Improving protein-protein interaction prediction using evolutionary information from low-quality MSAs.

PubMed

Várnai, Csilla; Burkoff, Nikolas S; Wild, David L

2017-01-01

Evolutionary information stored in multiple sequence alignments (MSAs) has been used to identify the interaction interface of protein complexes, by measuring either co-conservation or co-mutation of amino acid residues across the interface. Recently, maximum entropy related correlated mutation measures (CMMs) such as direct information, decoupling direct from indirect interactions, have been developed to identify residue pairs interacting across the protein complex interface. These studies have focussed on carefully selected protein complexes with large, good-quality MSAs. In this work, we study protein complexes with a more typical MSA consisting of fewer than 400 sequences, using a set of 79 intramolecular protein complexes. Using a maximum entropy based CMM at the residue level, we develop an interface level CMM score to be used in re-ranking docking decoys. We demonstrate that our interface level CMM score compares favourably to the complementarity trace score, an evolutionary information-based score measuring co-conservation, when combined with the number of interface residues, a knowledge-based potential and the variability score of individual amino acid sites. We also demonstrate, that, since co-mutation and co-complementarity in the MSA contain orthogonal information, the best prediction performance using evolutionary information can be achieved by combining the co-mutation information of the CMM with co-conservation information of a complementarity trace score, predicting a near-native structure as the top prediction for 41% of the dataset. The method presented is not restricted to small MSAs, and will likely improve interface prediction also for complexes with large and good-quality MSAs.

Stearoyl-Acyl Carrier Protein Desaturase Mutations Uncover an Impact of Stearic Acid in Leaf and Nodule Structure.

PubMed

Lakhssassi, Naoufal; Colantonio, Vincent; Flowers, Nicholas D; Zhou, Zhou; Henry, Jason; Liu, Shiming; Meksem, Khalid

2017-07-01

Stearoyl-acyl carrier protein desaturase (SACPD-C) has been reported to control the accumulation of seed stearic acid; however, no study has previously reported its involvement in leaf stearic acid content and impact on leaf structure and morphology. A subset of an ethyl methanesulfonate mutagenized population of soybean ( Glycine max ) 'Forrest' was screened to identify mutants within the GmSACPD-C gene. Using a forward genetics approach, one nonsense and four missense Gmsacpd-c mutants were identified to have high levels of seed, nodule, and leaf stearic acid content. Homology modeling and in silico analysis of the GmSACPD-C enzyme revealed that most of these mutations were localized near or at conserved residues essential for diiron ion coordination. Soybeans carrying Gmsacpd-c mutations at conserved residues showed the highest stearic acid content, and these mutations were found to have deleterious effects on nodule development and function. Interestingly, mutations at nonconserved residues show an increase in stearic acid content yet retain healthy nodules. Thus, random mutagenesis and mutational analysis allows for the achievement of high seed stearic acid content with no associated negative agronomic characteristics. Additionally, expression analysis demonstrates that nodule leghemoglobin transcripts were significantly more abundant in soybeans with deleterious mutations at conserved residues of GmSACPD-C. Finally, we report that Gmsacpd-c mutations cause an increase in leaf stearic acid content and an alteration of leaf structure and morphology in addition to differences in nitrogen-fixing nodule structure. © 2017 American Society of Plant Biologists. All Rights Reserved.

Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits.

PubMed Central

Kobayashi, H; Stewart, E; Poon, R; Adamczewski, J P; Gannon, J; Hunt, T

1992-01-01

The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase. Images PMID:1333843

Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits.

PubMed

Kobayashi, H; Stewart, E; Poon, R; Adamczewski, J P; Gannon, J; Hunt, T

1992-11-01

The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.

Voltage Sensing in Membranes: From Macroscopic Currents to Molecular Motions

PubMed Central

Freites, J. Alfredo; Tobias, Douglas J.

2015-01-01

Voltage-sensing domains (VSDs) are integral membrane protein units that sense changes in membrane electric potential, and through the resulting conformational changes, regulate a specific function. VSDs confer voltage-sensitivity to a large superfamily of membrane proteins that includes voltage-gated Na+, K+, Ca2+, and H+ selective channels, hyperpolarization-activated cyclic nucleotide-gated channels, and voltage-sensing phosphatases. VSDs consist of four transmembrane segments (termed S1 through S4). Their most salient structural feature is the highly conserved positions for charged residues in their sequences. S4 exhibits at least three conserved triplet repeats composed of one basic residue (mostly arginine) followed by two hydrophobic residues. These S4 basic side chains participate in a state-dependent internal salt-bridge network with at least four acidic residues in S1–S3. The signature of voltage-dependent activation in electrophysiology experiments is a transient current (termed gating or sensing current) upon a change in applied membrane potential as the basic side chains in S4 move across the membrane electric field. Thus, the unique structural features of the VSD architecture allow for competing requirements: maintaining a series of stable transmembrane conformations, while allowing charge motion, as briefly reviewed here. PMID:25972106

Determination of Multiple Near-Surface Residual Stress Components in Laser Peened Aluminum Alloy via the Contour Method

NASA Astrophysics Data System (ADS)

Toparli, M. Burak; Fitzpatrick, Michael E.; Gungor, Salih

2015-09-01

In this study, residual stress fields, including the near-surface residual stresses, were determined for an Al7050-T7451 sample after laser peening. The contour method was applied to measure one component of the residual stress, and the relaxed stresses on the cut surfaces were then measured by X-ray diffraction. This allowed calculation of the three orthogonal stress components using the superposition principle. The near-surface results were validated with results from incremental hole drilling and conventional X-ray diffraction. The results demonstrate that multiple residual stress components can be determined using a combination of the contour method and another technique. If the measured stress components are congruent with the principal stress axes in the sample, then this allows for determination of the complete stress tensor.

Steady-state fluorescence and phosphorescence spectroscopic studies of bacterial luciferase tryptophan mutants.

PubMed

Li, Z; Meighen, E A

1994-09-01

Bacterial luciferase, which catalyzes the bioluminescence reaction in luminous bacteria, consists of two nonidentical polypeptides, α and β. Eight mutants of luciferase with each of the tryptophans replaced by tyrosine were generated by site-directed mutagenesis and purified to homogeneity. The steady-state tryptophan fluorescence and low-temperature phosphorescence spectroscopic properties of these mutants were characterized. In some instances, mutation of only a single tryptophan residue resulted in large spectral changes. The tryptophan residues conserved in both the α and the β subunits exhibited distinct fluorescence emission properties, suggesting that these tryptophans have different local enviroments. The low-temperature phosphorescence data suggest that the tryptophans conserved in bot the α and the β subunits are not located at the subunit interface and/or involved in subunit interactions. The differences in the spectral properties of the mutants have provided useful information on the local environment of the individual tryptophan residues as well as on the quaternary structure of the protein.

Molecular basis of the activity of the phytopathogen pectin methylesterase

PubMed Central

Fries, Markus; Ihrig, Jessica; Brocklehurst, Keith; Shevchik, Vladimir E; Pickersgill, Richard W

2007-01-01

We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel β-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs. PMID:17717531

Fracture mechanics evaluation of heavy welded structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sprung, I.; Ericksson, C.W.; Zilberstein, V.A.

1982-05-01

This paper describes some applications of nondestructive examination (NDE) and engineering fracture mechanics to evaluation of flaws in heavy welded structures. The paper discusses not only widely recognized linear elastic fracture mechanics (LEFM) analysis, but also methods of the elastic-plastic fracture mechanics (EPFM), such as COD, J-integral, and Failure Assessment Diagram. Examples are given to highlight the importance of interaction between specialists providing input and the specialists performing the analysis. The paper points out that the critical parameters for as-welded structures when calculated by these methods are conservative since they are based on two pessimistic assumptions: that the magnitude ofmore » residual stress is always at the yield strength level, and that the residual stress always acts in the same direction as the applied (mechanical) stress. The suggestion is made that it would be prudent to use the COD or the FAD design curves for a conservative design. The appendix examines a J-design curve modified to include residual stresses.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

J Chang; S Xiang; K Xiang

The 5' {yields} 3' exoribonucleases (XRNs) have important functions in transcription, RNA metabolism and RNA interference. The structure of Rat1 (also known as Xrn2) showed that the two highly conserved regions of XRNs form a single, large domain that defines the active site of the enzyme. Xrn1 has a 510-residue segment after the conserved regions that is required for activity but is absent from Rat1/Xrn2. Here we report the crystal structures of Kluyveromyces lactis Xrn1 (residues 1-1,245, E178Q mutant), alone and in complex with a Mn{sup 2+} ion in the active site. The 510-residue segment contains four domains (D1-D4), locatedmore » far from the active site. Our mutagenesis and biochemical studies show that their functional importance results from their ability to stabilize the conformation of the N-terminal segment of Xrn1. These domains might also constitute a platform that interacts with protein partners of Xrn1.« less

A novel Arg H52/Tyr H33 conservative motif in antibodies: A correlation between sequence of antibodies and antigen binding.

PubMed

Petrov, Artem; Arzhanik, Vladimir; Makarov, Gennady; Koliasnikov, Oleg

2016-08-01

Antibodies are the family of proteins, which are responsible for antigen recognition. The computational modeling of interaction between an antigen and an antibody is very important when crystallographic structure is unavailable. In this research, we have discovered the correlation between the amino acid sequence of antibody and its specific binding characteristics on the example of the novel conservative binding motif, which consists of four residues: Arg H52, Tyr H33, Thr H59, and Glu H61. These residues are specifically oriented in the binding site and interact with each other in a specific manner. The residues of the binding motif are involved in interaction strictly with negatively charged groups of antigens, and form a binding complex. Mechanism of interaction and characteristics of the complex were also discovered. The results of this research can be used to increase the accuracy of computational antibody-antigen interaction modeling and for post-modeling quality control of the modeled structures.

Solution structure and DNA-binding properties of the C-terminal domain of UvrC from E.coli

PubMed Central

Singh, S.; Folkers, G.E.; Bonvin, A.M.J.J.; Boelens, R.; Wechselberger, R.; Niztayev, A.; Kaptein, R.

2002-01-01

The C-terminal domain of the UvrC protein (UvrC CTD) is essential for 5′ incision in the prokaryotic nucleotide excision repair process. We have determined the three-dimensional structure of the UvrC CTD using heteronuclear NMR techniques. The structure shows two helix–hairpin–helix (HhH) motifs connected by a small connector helix. The UvrC CTD is shown to mediate structure-specific DNA binding. The domain binds to a single-stranded–double-stranded junction DNA, with a strong specificity towards looped duplex DNA that contains at least six unpaired bases per loop (‘bubble DNA’). Using chemical shift perturbation experiments, the DNA-binding surface is mapped to the first hairpin region encompassing the conserved glycine–valine–glycine residues followed by lysine–arginine–arginine, a positively charged surface patch and the second hairpin region consisting of glycine–isoleucine–serine. A model for the protein– DNA complex is proposed that accounts for this specificity. PMID:12426397

High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation

PubMed Central

Anandapadamanaban, Madhanagopal; Andresen, Cecilia; Helander, Sara; Ohyama, Yoshifumi; Siponen, Marina I.; Lundström, Patrik; Kokubo, Tetsuro; Ikura, Mitsuhiko; Moche, Martin; Sunnerhagen, Maria

2016-01-01

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 when bound to yeast TBP, together with mutational data. The yTAF1-TAND1, which in itself acts as a transcriptional activator, binds into the DNA-binding TBP concave surface by presenting similar anchor residues to TBP as E. coli Mot1 but from a distinct structural scaffold. Furthermore, we show how yTAF1-TAND2 employs an aromatic and acidic anchoring pattern to bind a conserved yTBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides compelling insight into the competitive multiprotein TBP interplay critical to transcriptional regulation. PMID:23851461

High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation.

PubMed

Anandapadamanaban, Madhanagopal; Andresen, Cecilia; Helander, Sara; Ohyama, Yoshifumi; Siponen, Marina I; Lundström, Patrik; Kokubo, Tetsuro; Ikura, Mitsuhiko; Moche, Martin; Sunnerhagen, Maria

2013-08-01

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBP's concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

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

DOE PAGES

Teixeira, Miguel; Cabelli, Diane; Pinto, Ana F.; ...

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

Analysis of hepatitis B virus preS1 variability and prevalence of the rs2296651 polymorphism in a Spanish population

PubMed Central

Casillas, Rosario; Tabernero, David; Gregori, Josep; Belmonte, Irene; Cortese, Maria Francesca; González, Carolina; Riveiro-Barciela, Mar; López, Rosa Maria; Quer, Josep; Esteban, Rafael; Buti, Maria; Rodríguez-Frías, Francisco

2018-01-01

AIM To determine the variability/conservation of the domain of hepatitis B virus (HBV) preS1 region that interacts with sodium-taurocholate cotransporting polypeptide (hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism (S267F, NTCP variant) in a Spanish population. METHODS Serum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection (CHB) (n = 41, 73% Caucasians), patients with resolved HBV infection (n = 100, 100% Caucasians) and an HBV-uninfected control group (n = 105, 100% Caucasians). Variability/conservation of the amino acid (aa) sequences of the NTCP-interacting domain, (aa 2-48 in viral genotype D) and a highly conserved preS1 domain associated with virion morphogenesis (aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve analysis. RESULTS The HBV preS1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21 (in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCP-interacting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies (25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms (34% vs 27.3%), according to consensus sequences from GenBank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable (limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any participant. CONCLUSION In our CHB population, the NTCP-interacting domain was highly conserved, particularly the proline residues and essential amino acids related with the NTCP interaction, and the prevalence of rs2296651 was low/null. PMID:29456407

Effects of Zero Tillage (No-Till) Conservation Agriculture on soil physical and biological properties and their contributions to sustainability

NASA Astrophysics Data System (ADS)

Landers, John N.; Rass, Gerard; de Freitas, Pedro L.; Basch, Gottlieb; González Sanchez, Emilio J.; Tabaglio, Vincenzo; Kassan, Amir; Derpsch, Rolf; Friedrich, Theodor; Giupponi, Luca

2013-04-01

Not cultivating soil, rotating crops over the years, and leaving crop residues on the surface in the practice of zero tillage/conservation agriculture (ZT/CA) reverses the historically accelerating degradation of soil organic matter (SOM) and soil structure, while increasing soil biological activity by a factor of 2 to 4. The results of this are many: (a) not cultivating reduces soil compaction, leaving old root holes to facilitate internal drainage, averts the pulverization of soil aggregates and formation of pans, reduces draft power for planting and gives shelter, winter food and nesting sites for fauna, (b) crop residues on the surface practically eliminate wind and water erosion, reduce soil moisture loss through the mulch effect, slow spring warm-up (possibly offset by a lower specific heat demand with less water retention in surface soil) and act as a reserve of organically-compounded nutrients (as they decompose to humus), (c) more SOM means higher available water and nutrient retention, higher biological activity year round (enhancing biological controls), higher levels of water-stable aggregates and a positive carbon sink in incremental SOM. The positive impacts for society are: (i) more and cheaper food, (ii) reduced flood and drought-induced famine risks, (iii) a positive carbon sink in SOM and possible reductions in NO2 emissions, (iv) cleaner water and greater aquifer recharge due to reduced runoff, (v) cleaner air through effective elimination of dust as a product of cultivation (vi) less water pollution and greater aquifer recharge from reduced rainfall runoff, (vii) farm diesel consumption halved, (viii) reduced demand for (tropical) de-forestation, by permitting crop expansion on steeper lands, (ix) increased wildlife populations (skylarks, plovers, partridge and peccaries) and (x) an improved conservation mindset in farmers. It is notable that, in spite of successful practitioners in all European countries, mainstream adoption is still to come: Europés ZT/CA area is 1.35 million hectares, while the world area is now some 125 million and growing at a rate of 7 million hectares per year. More scientific measurements of the benefits of this system are required, both to assist adoption and to trigger policy measures. In the EEC, CAP reform (greening) needs to consider making environmental services payments for these social benefits since a reduction in single farm payments is ineluctable and carbon footprint reduction is of the essence, in the face of constantly-rising fuel prices and the need to cut GHG emissions. Therefore, as the principal farm tool which offers an effective and immediate solution towards positive changes in soil quality, productivity and sustainability, ZT/CA adoption needs financial incentives, which have high economic and environmental returns to society.

Alternative dimerization interfaces in the glucocorticoid receptor-α ligand binding domain.

PubMed

Bianchetti, Laurent; Wassmer, Bianca; Defosset, Audrey; Smertina, Anna; Tiberti, Marion L; Stote, Roland H; Dejaegere, Annick

2018-04-30

Nuclear hormone receptors (NRs) constitute a large family of multi-domain ligand-activated transcription factors. Dimerization is essential for their regulation, and both DNA binding domain (DBD) and ligand binding domain (LBD) are implicated in dimerization. Intriguingly, the glucocorticoid receptor-α (GRα) presents a DBD dimeric architecture similar to that of the homologous estrogen receptor-α (ERα), but an atypical dimeric architecture for the LBD. The physiological relevance of the proposed GRα LBD dimer is a subject of debate. We analyzed all GRα LBD homodimers observed in crystals using an energetic analysis based on the PISA and on the MM/PBSA methods and a sequence conservation analysis, using the ERα LBD dimer as a reference point. Several dimeric assemblies were observed for GRα LBD. The assembly generally taken to be physiologically relevant showed weak binding free energy and no significant residue conservation at the contact interface, while an alternative homodimer mediated by both helix 9 and C-terminal residues showed significant binding free energy and residue conservation. However, none of the GRα LBD assemblies found in crystals are as stable or conserved as the canonical ERα LBD dimer. GRα C-terminal sequence (F-domain) forms a steric obstacle to the canonical dimer assembly in all available structures. Our analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures. This work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research. Copyright © 2018. Published by Elsevier B.V.

Finite Element Analysis of Surface Residual Stress in Functionally Gradient Cemented Carbide Tool

NASA Astrophysics Data System (ADS)

Su, Chuangnan; Liu, Deshun; Tang, Siwen; Li, Pengnan; Qiu, Xinyi

2018-03-01

A component distribution model is proposed for three-component functionally gradient cemented carbide (FGCC) based on electron probe microanalysis results obtained for gradient layer thickness, microstructure, and elemental distribution. The residual surface stress of FGCC-T5 tools occurring during the fabrication process is analyzed using an ANSYS-implemented finite element method (FEM) and X-ray diffraction. A comparison of the experimental and calculated values verifies the feasibility of using FEM to analyze the residual surface stress in FGCC-T5 tools. The effects of the distribution index, geometrical shape, substrate thickness, gradient layer thickness, and position of the cobalt-rich layer on residual surface stress are studied in detail.

Adenocarcinoma in situ of the uterine cervix--a systematic review.

PubMed

Baalbergen, Astrid; Helmerhorst, Theo J M

2014-11-01

This study aimed to review literature if therapeutic strategies in adenocarcinoma in situ of the cervix could lead to a more conservative approach. A review of the literature was conducted using a Medline search for articles published between 1966 and 2013. Thirty-five studies showed that after a radical cone, 16.5% residual disease in the re-cone or uterus was found. After cone with positive margins, residual abnormalities were found in 49.3%. Thirty-seven studies showed 5% recurrence rate after conservative therapy (large loop excision transformation zone-cold knife conization. After conization with negative margins, the risk of recurrence was 3%. Adenocarcinoma in situ is a relatively rare premalignant but increasingly frequent lesion of the cervix. Although there is a risk of relapse (3%) with a chance of malignancy (<1%), this risk is so small that conservative treatment with negative margins by large loop excision transformation zone or cold knife conization is justified and justifiable not only for women to have children.

Crystal structure of a SLC11 (NRAMP) transporter reveals the basis for transition-metal ion transport.

PubMed

Ehrnstorfer, Ines A; Geertsma, Eric R; Pardon, Els; Steyaert, Jan; Dutzler, Raimund

2014-11-01

Members of the SLC11 (NRAMP) family transport iron and other transition-metal ions across cellular membranes. These membrane proteins are present in all kingdoms of life with a high degree of sequence conservation. To gain insight into the determinants of ion selectivity, we have determined the crystal structure of Staphylococcus capitis DMT (ScaDMT), a close prokaryotic homolog of the family. ScaDMT shows a familiar architecture that was previously identified in the amino acid permease LeuT. The protein adopts an inward-facing conformation with a substrate-binding site located in the center of the transporter. This site is composed of conserved residues, which coordinate Mn2+, Fe2+ and Cd2+ but not Ca2+. Mutations of interacting residues affect ion binding and transport in both ScaDMT and human DMT1. Our study thus reveals a conserved mechanism for transition-metal ion selectivity within the SLC11 family.

Molecular dynamics simulations of a K+ channel blocker: Tc1 toxin from Tityus cambridgei.

PubMed

Grottesi, Alessandro; Sansom, Mark S P

2003-01-30

Toxins that block voltage-gated potassium (Kv) channels provide a possible template for improved homology models of the Kv pore. In assessing the interactions of Kv channels and their toxins it is important to determine the dynamic flexibility of the toxins. Multiple 10 ns duration molecular dynamics simulations combined with essential dynamics analysis have been used to explore the flexibility of four different Kv channel-blocking toxins. Three toxins (Tc1, AgTx and ChTx) share a common fold. They also share a common pattern of conformational dynamics, as revealed by essential dynamics analysis of the simulation results. This suggests that some aspects of dynamic behaviour are conserved across a single protein fold class. In each of these three toxins, the residue exhibiting minimum flexibility corresponds to a conserved lysine residue that is suggested to interact with the filter domain of the channel. Thus, comparative simulations reveal functionally important conservation of molecular dynamics as well as protein fold across a family of related toxins.

PESTICIDE SURFACE RESIDUE MEASUREMENTS BY A PRESS SAMPLER

EPA Science Inventory

Pesticides on household surfaces are a source of exposure to children. Accurate measurements of residues on surfaces are needed to determine amounts available for transfer to foods and other objects handled or eaten by a child. Wiping the surface with a solvent has been the acc...

Weed management and cotton yield under two row spacings, conventional and conservation tillage systems utilizing conventional, glufosinate-, and glyphosate-based weed management systems

USDA-ARS?s Scientific Manuscript database

A field experiment was conducted in 2005 and 2006, to evaluate weed control in conventional, Liberty Link® (LL), and Roundup Ready® (RR) herbicide systems under standard [102 cm (40 inch)] and narrow [38 cm (15 inch)] row-spacings utilizing conventional and high-residue conservation tillage systems....

Flight Testing Surfaces Engineered for Mitigating Insect Adhesion on a Falcon HU-25C

NASA Technical Reports Server (NTRS)

Shanahan, Michelle; Wohl, Chris J.; Smith, Joseph G., Jr.; Connell, John W.; Siochi, Emilie J.; Doss, Jereme R.; Penner, Ronald K.

2015-01-01

Insect residue contamination on aircraft wings can decrease fuel efficiency in aircraft designed for natural laminar flow. Insect residues can cause a premature transition to turbulent flow, increasing fuel burn and making the aircraft less environmentally friendly. Surfaces, designed to minimize insect residue adhesion, were evaluated through flight testing on a Falcon HU-25C aircraft flown along the coast of Virginia and North Carolina. The surfaces were affixed to the wing leading edge and the aircraft remained at altitudes lower than 1000 feet throughout the flight to assure high insect density. The number of strikes on the engineered surfaces was compared to, and found to be lower than, untreated aluminum control surfaces flown concurrently. Optical profilometry was used to determine insect residue height and areal coverage. Differences in results between flight and laboratory tests suggest the importance of testing in realistic use environments to evaluate the effectiveness of engineered surface designs.

Structural basis for corepressor assembly by the orphan nuclear receptor TLX

PubMed Central

Zhou, X. Edward; He, Yuanzheng; Searose-Xu, Kelvin; Zhang, Chun-Li; Tsai, Chih-Cheng; Melcher, Karsten

2015-01-01

The orphan nuclear receptor TLX regulates neural stem cell self-renewal in the adult brain and functions primarily as a transcription repressor through recruitment of Atrophin corepressors, which bind to TLX via a conserved peptide motif termed the Atro box. Here we report crystal structures of the human and insect TLX ligand-binding domain in complex with Atro box peptides. In these structures, TLX adopts an autorepressed conformation in which its helix H12 occupies the coactivator-binding groove. Unexpectedly, H12 in this autorepressed conformation forms a novel binding pocket with residues from helix H3 that accommodates a short helix formed by the conserved ALXXLXXY motif of the Atro box. Mutations that weaken the TLX–Atrophin interaction compromise the repressive activity of TLX, demonstrating that this interaction is required for Atrophin to confer repressor activity to TLX. Moreover, the autorepressed conformation is conserved in the repressor class of orphan nuclear receptors, and mutations of corresponding residues in other members of this class of receptors diminish their repressor activities. Together, our results establish the functional conservation of the autorepressed conformation and define a key sequence motif in the Atro box that is essential for TLX-mediated repression. PMID:25691470

Development by Design in Colombia: Making Mitigation Decisions Consistent with Conservation Outcomes

PubMed Central

Saenz, Shirley; Walschburger, Tomas; González, Juan Carlos; León, Jorge; McKenney, Bruce; Kiesecker, Joseph

2013-01-01

Mitigation policy and regulatory frameworks are consistent in their strong support for the mitigation hierarchy of: (1) avoiding impacts, (2) minimizing impacts, and then (3) offsetting/compensating for residual impacts. While mitigation frameworks require developers to avoid, minimize and restore biodiversity on-site before considering an offset for residual impacts, there is a lack of quantitative guidance for this decision-making process. What are the criteria for requiring impacts be avoided altogether? Here we examine how conservation planning can guide the application of the mitigation hierarchy to address this issue. In support of the Colombian government's aim to improve siting and mitigation practices for planned development, we examined five pilot projects in landscapes expected to experience significant increases in mining, petroleum and/or infrastructure development. By blending landscape-level conservation planning with application of the mitigation hierarchy, we can proactively identify where proposed development and conservation priorities would be in conflict and where impacts should be avoided. The approach we outline here has been adopted by the Colombian Ministry of Environment and Sustainable Development to guide licensing decisions, avoid piecemeal licensing, and promote mitigation decisions that maintain landscape condition. PMID:24339972

Development by design in Colombia: making mitigation decisions consistent with conservation outcomes.

PubMed

Saenz, Shirley; Walschburger, Tomas; González, Juan Carlos; León, Jorge; McKenney, Bruce; Kiesecker, Joseph

2013-01-01

Mitigation policy and regulatory frameworks are consistent in their strong support for the mitigation hierarchy of: (1) avoiding impacts, (2) minimizing impacts, and then (3) offsetting/compensating for residual impacts. While mitigation frameworks require developers to avoid, minimize and restore biodiversity on-site before considering an offset for residual impacts, there is a lack of quantitative guidance for this decision-making process. What are the criteria for requiring impacts be avoided altogether? Here we examine how conservation planning can guide the application of the mitigation hierarchy to address this issue. In support of the Colombian government's aim to improve siting and mitigation practices for planned development, we examined five pilot projects in landscapes expected to experience significant increases in mining, petroleum and/or infrastructure development. By blending landscape-level conservation planning with application of the mitigation hierarchy, we can proactively identify where proposed development and conservation priorities would be in conflict and where impacts should be avoided. The approach we outline here has been adopted by the Colombian Ministry of Environment and Sustainable Development to guide licensing decisions, avoid piecemeal licensing, and promote mitigation decisions that maintain landscape condition.

Improving homology modeling of G-protein coupled receptors through multiple-template derived conserved inter-residue interactions

NASA Astrophysics Data System (ADS)

Chaudhari, Rajan; Heim, Andrew J.; Li, Zhijun

2015-05-01

Evidenced by the three-rounds of G-protein coupled receptors (GPCR) Dock competitions, improving homology modeling methods of helical transmembrane proteins including the GPCRs, based on templates of low sequence identity, remains an eminent challenge. Current approaches addressing this challenge adopt the philosophy of "modeling first, refinement next". In the present work, we developed an alternative modeling approach through the novel application of available multiple templates. First, conserved inter-residue interactions are derived from each additional template through conservation analysis of each template-target pairwise alignment. Then, these interactions are converted into distance restraints and incorporated in the homology modeling process. This approach was applied to modeling of the human β2 adrenergic receptor using the bovin rhodopsin and the human protease-activated receptor 1 as templates and improved model quality was demonstrated compared to the homology model generated by standard single-template and multiple-template methods. This method of "refined restraints first, modeling next", provides a fast and complementary way to the current modeling approaches. It allows rational identification and implementation of additional conserved distance restraints extracted from multiple templates and/or experimental data, and has the potential to be applicable to modeling of all helical transmembrane proteins.

The influence of alloy composition on residual stresses in heat treated aluminium alloys

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robinson, J.S., E-mail: jeremy.robinson@ul.ie; Redington, W.

The as quenched properties of eight different heat treatable aluminium alloys are related to residual stress magnitudes with the objective being to establish if there is a relationship between the residual stress and the as quenched alloy hardness and strength. Near surface residual stresses were assessed with X-ray diffraction using both the established sin{sup 2}ψ method and the more recent cos α technique. Through thickness residual stresses were also characterised using neutron diffraction. The alloys were chosen to encompass a wide range of strengths. The low to medium strength alloys were 6060 and 6082, medium to high strength 2618A, 2014A,more » 7075, 7010 and two variants of 7449, while the very high strength alloy was the powder metallurgy alloy N707. To assess the as quenched strength, dynamic hardness and tensile properties were determined from samples tested immediately after quenching to minimise the influence of precipitation hardening by natural aging. In addition, hot hardness measurements were made in situ on samples cooled to simulate quench paths. Within the experimental constraints of the investigation, the distribution of residual stress through the thickness was found to follow the same pattern for all the alloys investigated, varying from tensile in the interior to surface compression. The influence of alloy strength was manifested as a change in the observed residual stress magnitudes, and surface residual stresses were found to vary linearly with as quenched hardness and strength. - Highlights: • As quenched aluminium alloys contain high magnitude residual stresses. • Surface is compressive balance by a tensile core. • As quenched surface residual stress is linear function of alloy strength. • In situ hot hardness demonstrates rapid change in intrinsic hardness during rapid cooling.« less

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks

PubMed Central

Patil, Ashwini; Nakamura, Haruki

2007-01-01

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long – range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions. PMID:27857564

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks.

PubMed

Patil, Ashwini; Nakamura, Haruki

2007-01-01

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long - range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions.

Insect Residue Contamination on Wing Leading Edge Surfaces: A Materials Investigation for Mitigation

NASA Technical Reports Server (NTRS)

Lorenzi, Tyler M.; Wohl, Christopher J.; Penner, Ronald K.; Smith, Joseph G.; Siochi, Emilie J.

2011-01-01

Flight tests have shown that residue from insect strikes on aircraft wing leading edge surfaces may induce localized transition of laminar to turbulent flow. The highest density of insect populations have been observed between ground level and 153 m during light winds (2.6 -- 5.1 m/s), high humidity, and temperatures from 21 -- 29 C. At a critical residue height, dependent on the airfoil and Reynolds number, boundary layer transition from laminar to turbulent results in increased drag and fuel consumption. Although this represents a minimal increase in fuel burn for conventional transport aircraft, future aircraft designs will rely on maintaining laminar flow across a larger portion of wing surfaces to reduce fuel burn during cruise. Thus, insect residue adhesion mitigation is most critical during takeoff and initial climb to maintain laminar flow in fuel-efficient aircraft configurations. Several exterior treatments investigated to mitigate insect residue buildup (e.g., paper, scrapers, surfactants, flexible surfaces) have shown potential; however, implementation has proven to be impractical. Current research is focused on evaluation of wing leading edge surface coatings that may reduce insect residue adhesion. Initial work under NASA's Environmentally Responsible Aviation Program focused on evaluation of several commercially available products (commercial off-the-shelf, COTS), polymers, and substituted alkoxy silanes that were applied to aluminum (Al) substrates. Surface energies of these coatings were determined from contact angle data and were correlated to residual insect excrescence on coated aluminum substrates using a custom-built "bug gun." Quantification of insect excrescence surface coverage was evaluated by a series of digital photographic image processing techniques.

Non-destructive measurement and role of surface residual stress monitoring in residual life assessment of a steam turbine blading material

NASA Astrophysics Data System (ADS)

Prabhu-Gaunkar, Gajanana; Rawat, M. S.; Prasad, C. R.

2014-02-01

Steam turbine blades in power generation equipment are made from martensitic stainless steels having high strength, good toughness and corrosion resistance. However, these steels are susceptible to pitting which can promote early failures of blades in the turbines, particularly in the low pressure dry/wet areas by stress corrosion and corrosion fatigue. Presence of tensile residual stresses is known to accelerate failures whereas compressive stresses can help in delaying failures. Shot peening has been employed as an effective tool to induce compressive residual stresses which offset a part of local surface tensile stresses in the surface layers of components. Maintaining local stresses at stress raisers, such as pits formed during service, below a threshold level can help in preventing the initiation microcracks and failures. The thickness of the layer in compression will, however, depend of the shot peening parameters and should extend below the bottom of corrosion pits. The magnitude of surface compressive drops progressively during service exposure and over time the effectiveness of shot peening is lost making the material susceptible to micro-crack initiation once again. Measurement and monitoring of surface residual stress therefore becomes important for assessing residual life of components in service. This paper shows the applicability of surface stress monitoring to life assessment of steam turbine blade material based on data generated in laboratory on residual surface stress measurements in relation to fatigue exposure. An empirical model is proposed to calculate the remaining life of shot peened steam turbine blades in service.

Modelling the dissipation and leaching of two herbicides in decomposing mulch of crop residues

NASA Astrophysics Data System (ADS)

Aslam, Sohaib; Iqbal, Akhtar; Lafolie, François; Recous, Sylvie; Benoit, Pierre; Garnier, Patricia

2013-04-01

Conservation agricultural practices are increasingly adopted because of ecosystem services such as conservation of soil and water resources. These farming systems are characterized mainly by the presence of mulch made of residues of harvested or cover crops on soil surface. The mulch can intercept and retain applied pesticides depending on pesticide molecule and rainfall timing. The pesticide wash-off from mulch is considered a key process in pesticide fate and can have effects on degradation and transport processes. This work highlights a modelling approach to study the pesticide wash-off from mulch residues and their further transport in soil under two rainfall regimes. Transformation and leaching of two herbicides, s-metolachlor and glyphosate, was studied and simulated by Pastis-mulch model. A pesticide module describing pesticide degradation in mulch and soil was coupled to a transport model including a mulch module. The model was tested to simulate the pesticide dissipation, wash-off from mulch and further leaching in soil. Pesticide degradation parameters in mulch were estimated from incubation experiments with 14C-labelled molecules in small cylinders. The model was then tested using the data obtained through a soil column experiment (reconstructed soil cores :15 cm diameter x 35 cm depth), a mulch of Zea mais + Doliquos lablab and with two treatments varied by water regimes: i) frequent rain (temperate, twice a week) with week intensity (6 mm/hr); and ii) occasional rain (tropical, twice a month) with stronger intensity (20 mm/hr). Columns were incubated at 20 °C for 84 days to monitor soil water, C, N and pesticide dynamics. Model successfully simulated the experimental data of pesticide dissipation in mulch residues. Results showed that the rain regime affected more S-metolachlor than glyphosate behavior. The simulated results indicated also that the dynamics in mulch of the two molecules differed according to the rain treatment. Glyphosate showed a greater leaching from mulch than S-metolachlor because of its lower adsorption coefficients to organic mulch. Moreover, simulated results showed a much faster degradation of glyphosate but greater non-extractable residue formation for S-metolachlor. Keywords: Mulch; Pesticides; Transport; Degradation; Modeling; Pastis-mulch References Findeling, A., Garnier, P., Coppens, F., Lafolie, F., Recous, S., 2007. Modelling water, carbon and nitrogen dynamics in soil covered with decomposing mulch. European Journal of Soil Science 58, 196-206. Lashermes, G., Zhang, Y., Houot, S., Barriuso, E., Steyer, J.P., Patureau, D., Garnier, P., 2013. A model coupling organic carbon and organic pollutant dynamics during composting. Journal of Environmental Quality. In Press.

Evolution of residual stress, free volume, and hardness in the laser shock peened Ti-based metallic glass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Liang; Wang, Lu; Nie, Zhihua

Laser shock peening (LSP) with different cycles was performed on the Ti-based bulk metallic glasses (BMGs). The sub-surface residual stress of the LSPed specimens was measured by high-energy X-ray diffraction (HEXRD) and the near-surface residual stress was measured by scanning electron microscope/focused ion beam (SEM/FIB) instrument. The sub-surface residual stress in the LSP impact direction (about-170MPa) is much lower than that perpendicular to the impact direction (about -350 MPa), exhibiting anisotropy. The depth of the compressive stress zone increases from 400 mu m to 500 mu m with increasing LSP cycles. The highest near-surface residual stress is about -750 MPa.more » LSP caused the free volume to increase and the maximum increase appeared after the first LSP process. Compared with the hardness (567 +/- 7 HV) of the as-cast BMG, the hardness (590 +/- 9 HV) on the shocked surface shows a hardening effect due to the hardening mechanism of compressive residual stress; and the hardness (420 +/- 9 HV) on the longitudinal section shows a softening effect due to the softening mechanism of free volume.« less

TRANSFER OF PESTICIDES FROM SURFACES TO FOODS FOR THE ESTIMATION OF DIETARY EXPOSURE OF CHILDREN

EPA Science Inventory

Since small children spend much of their time in contact with contaminated surfaces, residues of pesticides found on floors and other surfaces contribute to their aggregate exposures. Any dislodgeable pesticide residues on hands and surfaces may be transferred to foods while bei...

Analysis by mutagenesis of the ATP binding site of the gamma subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system.

PubMed

Lee, J H; Maeda, S; Angelos, K L; Kamita, S G; Ramachandran, C; Walsh, D A

1992-11-03

Active gamma subunit of skeletal muscle phosphorylase kinase has been obtained by expression of the rat soleus cDNA in a baculovirus system. The protein exhibited the expected pH 6.8/8.2 activity ratio of 0.6, and its activity was insensitive to Ca2+ addition, indicating that it was free gamma subunit and not a gamma subunit-calmodulin complex. It was stimulated approximately 2-fold by Ca(2+)-calmodulin addition, demonstrating that it had retained high-affinity calmodulin binding. By site-directed mutagenesis, we have examined the role of six of the amino acids that constitute the consensus ATP binding site of the protein kinase, which in the gamma subunit is represented by the sequence 26Gly.Arg.Gly.Val.Ser.Ser.Val.Val33. Changes were evaluated by the kinetic determination of the dissociation constants of gamma-ATP, gamma-ADP, gamma-AMP.PCP, and gamma-phosphorylase and the maximum catalytic activity. The mutants Ser26-gamma, Ser29-gamma, Phe30-gamma, and Gly31-gamma each exhibited an essentially identical dissociation constant for gamma subunit phosphorylase, indicating that these mutations had not caused a global alteration in the protein structure but were limited to changes in the nucleotide binding site domain. Substitution of either Val33 (by Gly) or Gly28 (by Ser), two of the most conserved residues in all protein kinases, resulted in enzyme with marginally detectable activity. In noted contrast, the Ser26 mutant, which substituted the first glycine of the consensus glycine trio motif, and which is also very highly conserved, retained at least 25% of the enzymatic activity. The Gly31 substitution, which restored a glycine to a position characteristic for most protein kinases, had little overall effect upon the maximum rate of catalysis. Restoration of Ser30 to the more typical phenylalanine, which is present in most protein kinases, had minimal effect on catalysis. These data provide the first direct evaluation of the roles that different residues play within this consensus glycine trio/valine motif of the protein kinases, which up to now have only been surmised to be of importance because of their conservation. Two unexpected findings are that for one residue that is very conserved (Gly26) there is some flexibility of substitution not apparent from the evolutionary conservation and that a second quite conserved residue in protein kinases (equivalent to Gly at position 31) does not produce a protein optimized for nucleotide binding.

Charge requirements of lipid II flippase activity in Escherichia coli.

PubMed

Butler, Emily K; Tan, Wee Boon; Joseph, Hildy; Ruiz, Natividad

2014-12-01

Peptidoglycan (PG) is an extracytoplasmic glycopeptide matrix essential for the integrity of the envelope of most bacteria. The PG building block is a disaccharide-pentapeptide that is synthesized as a lipid-linked precursor called lipid II. The translocation of the amphipathic lipid II across the cytoplasmic membrane is required for subsequent incorporation of the disaccharide-pentapeptide into PG. In Escherichia coli, the essential inner membrane protein MurJ is the lipid II flippase. Previous studies showed that 8 charged residues in the central cavity region of MurJ are crucial for function. Here, we completed the functional analysis of all 57 charged residues in MurJ and demonstrated that the respective positive or negative charge of the 8 aforementioned residues is required for proper MurJ function. Loss of the negative charge in one of these residues, D39, causes a severe defect in MurJ biogenesis; by engineering an intragenic suppressor mutation that restores MurJ biogenesis, we found that this charge is also essential for MurJ function. Because of the low level of homology between MurJ and putative orthologs from Gram-positive bacteria, we explored the conservation of these 8 charged residues in YtgP, a homolog from Streptococcus pyogenes. We found that only 3 positive charges are similarly positioned and essential in YtgP; YtgP possesses additional charged residues within its predicted cavity that are essential for function and conserved among Gram-positive bacteria. From these data, we hypothesize that some charged residues in the cavity region of MurJ homologs are required for interaction with lipid II and/or energy coupling during transport. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

On inter-tidal transport equation

USGS Publications Warehouse

Cheng, Ralph T.; Feng, Shizuo; Pangen, Xi

1989-01-01

The transports of solutes, sediments, nutrients, and other tracers are fundamental to the interactive physical, chemical, and biological processes in estuaries. The characteristic time scales for most estuarine biological and chemical processes are on the order of several tidal cycles or longer. To address the long-term transport mechanism meaningfully, the formulation of an inter-tidal conservation equation is the main subject of this paper. The commonly used inter-tidal conservation equation takes the form of a convection-dispersion equation in which the convection is represented by the Eulerian residual current, and the dispersion terms are due to the introduction of a Fickian hypothesis, unfortunately, the physical significance of this equation is not clear, and the introduction of a Fickian hypothesis is at best an ad hoc approximation. Some recent research results on the Lagrangian residual current suggest that the long-term transport problem is more closely related to the Lagrangian residual current than to the Eulerian residual current. With the aid of additional insight of residual current, the inter-tidal transport equation has been reformulated in this paper using a small perturbation method for a weakly nonlinear tidal system. When tidal flows can be represented by an M2 system, the new intertidal transport equation also takes the form of a convective-dispersion equation without the introduction of a Fickian hypothesis. The convective velocity turns out to be the first order Lagrangian residual current (the sum of the Eulerian residual current and the Stokes’ drift), and the correlation terms take the form of convection with the Stokes’ drift as the convective velocity. The remaining dispersion terms are perturbations of lower order solution to higher order solutions due to shear effect and turbulent mixing.

Quantitative expression of protein heterogeneity: Response of amino acid side chains to their local environment.

PubMed

Bandyopadhyay, Debashree; Mehler, Ernest L

2008-08-01

A general method has been developed to characterize the hydrophobicity or hydrophilicity of the microenvironment (MENV), in which a given amino acid side chain is immersed, by calculating a quantitative property descriptor (QPD) based on the relative (to water) hydrophobicity of the MENV. Values of the QPD were calculated for a test set of 733 proteins to analyze the modulating effects on amino acid residue properties by the MENV in which they are imbedded. The QPD values and solvent accessibility were used to derive a partitioning of residues based on the MENV hydrophobicities. From this partitioning, a new hydrophobicity scale was developed, entirely in the context of protein structure, where amino acid residues are immersed in one or more "MENVpockets." Thus, the partitioning is based on the residues "sampling" a large number of "solvents" (MENVs) that represent a very large range of hydrophobicity values. It was found that the hydrophobicity of around 80% of amino acid side chains and their MENV are complementary to each other, but for about 20%, the MENV and their imbedded residue can be considered as mismatched. Many of these mismatches could be rationalized in terms of the structural stability of the protein and/or the involvement of the imbedded residue in function. The analysis also indicated a remarkable conservation of local environments around highly conserved active site residues that have similar functions across protein families, but where members have relatively low sequence homology. Thus, quantitative evaluation of this QPD is suggested, here, as a tool for structure-function prediction, analysis, and parameter development for the calculation of properties in proteins. (c) 2008 Wiley-Liss, Inc.

Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2-MPER region.

PubMed

Flyak, Andrew I; Kuzmina, Natalia; Murin, Charles D; Bryan, Christopher; Davidson, Edgar; Gilchuk, Pavlo; Gulka, Christopher P; Ilinykh, Philipp A; Shen, Xiaoli; Huang, Kai; Ramanathan, Palaniappan; Turner, Hannah; Fusco, Marnie L; Lampley, Rebecca; Kose, Nurgun; King, Hannah; Sapparapu, Gopal; Doranz, Benjamin J; Ksiazek, Thomas G; Wright, David W; Saphire, Erica Ollmann; Ward, Andrew B; Bukreyev, Alexander; Crowe, James E

2018-05-07

Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMapp, which has been shown to be effective in nonhuman primate models of infection 1 and has been used under compassionate-treatment protocols in humans 2 . ZMapp is a mixture of three chimerized murine monoclonal antibodies (mAbs) 3-6 that target EBOV-specific epitopes on the surface glycoprotein 7,8 . However, ZMapp mAbs do not neutralize other species from the genus Ebolavirus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here, we describe three naturally occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane-proximal external region (MPER) of the glycoprotein. The identification of a conserved neutralizing antigenic site in the glycoprotein suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2-MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.

In silico modeling of the Moniliophthora perniciosa Atg8 protein.

PubMed

Pereira, A C F; Cardoso, T H S; Brendel, M; Pungartnik, C

2013-12-11

Autophagy is defined as an intracellular system of lysosomal degradation in eukaryotic cells, and the genes involved in this process are conserved from yeast to humans. Among these genes, ATG8 encodes a ubiquitin-like protein that is conjugated to a phosphatidylethanolamine (PE) membrane by the ubiquitination system. The Atg8p-PE complex is important in initiating the formation of the autophagosome and thus plays a critical role in autophagy. In silico modeling of Atg8p of Moniliophthora perniciosa revealed its three-dimensional structure and enabled comparison with its Saccharomyces cerevisiae homologue ScAtg8p. Some common and distinct features were observed between these two proteins, including the conservation of residues required to allow the interaction of α-helix1 with the ubiquitin core. However, the electrostatic potential surfaces of these helices differ, implying particular roles in selecting specific binding partners. The proposed structure was validated by the programs PROCHECK 3.4, ANOLEA, and QMEAN, which demonstrated 100% of amino acids located in favorable regions with low total energy. Our results showed that MpAtg8p contains the same functional domains (3 α-helices and 4 β-sheets) and is similar in structure as the ScAtg8p yeast. Both proteins have many conserved sequences in common, and therefore, their proposed three-dimensional models show similar configuration.

Highly conserved small subunit residues influence rubisco large subunit catalysis.

PubMed

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

PubMed Central

Coopman, K.; Wallis, R.; Robb, G.; Brown, A. J. H.; Wilkinson, G. F.; Timms, D.

2011-01-01

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues. PMID:21868452

Molecular docking and simulation studies of gustatory receptor of Aedes aegypti: A potent drug target to distract host-seeking behaviour in mosquitoes.

PubMed

Gupta, Krishna Kant; Sethi, Guneswar; Jayaraman, Manikandan

2016-01-01

It is well reported that exhaled CO 2 and skin odour from human being assist female mosquitoes to locate human host. Basically, the receptors for this activity are expressed in cpA neurons. In both Aedes aegypti and Anopheles gambiae, this CO 2-sensitive olfactory neuron detects myriad number of chemicals present in human skin. Therefore, manipulation of gustatory receptors housing these neurons may serve as important targets for behavioural intervention. The study was aimed towards virtual screening of small molecules in the analyzed conserved active site residues of gustatory receptor and molecular dynamics simulation study of optimum protein-ligand complex to identify a suitable lead molecule for distracting host-seeking behaviour of mosquitoes. The conserved residue analysis of gustatory receptor (GR) of Ae. aegypti and An. gambiae was performed. The structure of GR protein from Ae. aegypti was modeled and validated, and then molecular docking was performed to screen 2903 small molecules against the predicted active residues of GR. Further, simulation studies were also carried out to prove protein-ligand stability. The glutamine 154 residue of GR was found to be highly conserved in Ae. aegypti and An. gambiae. Docking results indicated that the dodecanoic acid, 1,2,3-propanetriyl ester (dynasan 112) was interacting with this residue, as it showed better LibDock score than previously reported ethyl acetate used as mosquito repellant. Simulation studies indicated the structural instability of GR protein in docked form with dynasan 112 suggesting its involvement in structural changes. Based on the interaction energies and stability, this compound has been proposed to be used in mosquitoes' repellant. A novel effective odorant acting as inhibitor of GR is proposed based on its stability, docking score, interactions and RMSD, considering ethyl pyruvate as a standard inhibitor. Host preference and host-seeking ability of mosquito vectors play key roles in disease transmission, a clear understanding of these aspects is essential for preventing the spread of the disease.

Engineered Surfaces for Mitigation of Insect Residue Adhesion

NASA Technical Reports Server (NTRS)

Siochi, Emilie J.; Smith, Joseph G.; Wohl, Christopher J.; Gardner, J. M.; Penner, Ronald K.; Connell, John W.

2013-01-01

Maintenance of laminar flow under operational flight conditions is being investigated under NASA s Environmentally Responsible Aviation (ERA) Program. Among the challenges with natural laminar flow is the accretion of residues from insect impacts incurred during takeoff or landing. Depending on air speed, temperature, and wing structure, the critical residue height for laminar flow disruption can be as low as 4 microns near the leading edge. In this study, engineered surfaces designed to minimize insect residue adhesion were examined. The coatings studied included chemical compositions containing functional groups typically associated with abhesive (non-stick) surfaces. To reduce surface contact by liquids and enhance abhesion, the engineered surfaces consisted of these coatings doped with particulate additives to generate random surface topography, as well as coatings applied to laser ablated surfaces having precision patterned topographies. Performance evaluation of these surfaces included contact angle goniometry of pristine coatings and profilometry of surfaces after insect impacts were incurred in laboratory scale tests, wind tunnel tests and flight tests. The results illustrate the complexity of designing antifouling surfaces for effective insect contamination mitigation under dynamic conditions and suggest that superhydrophobic surfaces may not be the most effective solution for preventing insect contamination on aircraft wing leading edges.

Effects of Ultrasonic Nanocrystal Surface Modification on the Residual Stress, Microstructure, and Corrosion Resistance of 304 Stainless Steel Welds

NASA Astrophysics Data System (ADS)

Ye, Chang; Telang, Abhishek; Gill, Amrinder; Wen, Xingshuo; Mannava, Seetha R.; Qian, Dong; Vasudevan, Vijay K.

2018-03-01

In this study, ultrasonic nanocrystal surface modification (UNSM) of 304 stainless steel welds was carried out. UNSM effectively eliminates the tensile stress generated during welding and imparts beneficial compressive residual stresses. In addition, UNSM can effectively refine the grains and increase hardness in the near-surface region. Corrosion tests in boiling MgCl2 solution demonstrate that UNSM can significantly improve the corrosion resistance due to the compressive residual stresses and changes in the near-surface microstructure.

InterProSurf: a web server for predicting interacting sites on protein surfaces

PubMed Central

Negi, Surendra S.; Schein, Catherine H.; Oezguen, Numan; Power, Trevor D.; Braun, Werner

2009-01-01

Summary A new web server, InterProSurf, predicts interacting amino acid residues in proteins that are most likely to interact with other proteins, given the 3D structures of subunits of a protein complex. The prediction method is based on solvent accessible surface area of residues in the isolated subunits, a propensity scale for interface residues and a clustering algorithm to identify surface regions with residues of high interface propensities. Here we illustrate the application of InterProSurf to determine which areas of Bacillus anthracis toxins and measles virus hemagglutinin protein interact with their respective cell surface receptors. The computationally predicted regions overlap with those regions previously identified as interface regions by sequence analysis and mutagenesis experiments. PMID:17933856

The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii.

PubMed Central

Sridhar Prasad, G.; Kresge, N.; Muhlberg, A. B.; Shaw, A.; Jung, Y. S.; Burgess, B. K.; Stout, C. D.

1998-01-01

NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif. The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258. PMID:9865948

Sequence analysis of serum albumins reveals the molecular evolution of ligand recognition properties.

PubMed

Fanali, Gabriella; Ascenzi, Paolo; Bernardi, Giorgio; Fasano, Mauro

2012-01-01

Serum albumin (SA) is a circulating protein providing a depot and carrier for many endogenous and exogenous compounds. At least seven major binding sites have been identified by structural and functional investigations mainly in human SA. SA is conserved in vertebrates, with at least 49 entries in protein sequence databases. The multiple sequence analysis of this set of entries leads to the definition of a cladistic tree for the molecular evolution of SA orthologs in vertebrates, thus showing the clustering of the considered species, with lamprey SAs (Lethenteron japonicum and Petromyzon marinus) in a separate outgroup. Sequence analysis aimed at searching conserved domains revealed that most SA sequences are made up by three repeated domains (about 600 residues), as extensively characterized for human SA. On the contrary, lamprey SAs are giant proteins (about 1400 residues) comprising seven repeated domains. The phylogenetic analysis of the SA family reveals a stringent correlation with the taxonomic classification of the species available in sequence databases. A focused inspection of the sequences of ligand binding sites in SA revealed that in all sites most residues involved in ligand binding are conserved, although the versatility towards different ligands could be peculiar of higher organisms. Moreover, the analysis of molecular links between the different sites suggests that allosteric modulation mechanisms could be restricted to higher vertebrates.

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats.

PubMed

de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

2015-11-16

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Conserved Aromatic Residue Confers Cation Selectivity in Claudin-2 and Claudin-10b*

PubMed Central

Li, Jiahua; Zhuo, Min; Pei, Lei; Yu, Alan S. L.

2013-01-01

In tight junctions, both claudin-2 and claudin-10b form paracellular cation-selective pores by the interaction of the first ECL 1 with permeating ions. We hypothesized that a highly conserved aromatic residue near the pore selectivity filter of claudins contributes to cation selectivity by cation-π interaction with the permeating cation. To test this, we generated MDCK I Tet-off cells stably transfected with claudin-2 Tyr67 mutants. The Y67L mutant showed reduced cation selectivity compared with wild-type claudin-2 due to a decrease in Na+ permeability, without affecting the Cl− permeability. The Y67A mutant enlarged the pore size and further decreased the charge selectivity due to an increase in Cl− permeability. The Y67F mutant restored the Na+ permeability, Cl− permeability, and pore size back to wild-type. The accessibility of Y67C to methanethiosulfonate modification indicated that its side chain faces the lumen of the pore. In claudin-10b, the F66L mutant reduced cation selectivity, and the F66A mutant lost pore conductance. We conclude that the conserved aromatic residue near the cation pore domain of claudins contributes to cation selectivity by a dual role of cation-π interaction and a luminal steric effect. Our findings provide new insight into how ion selectivity is achieved in the paracellular pore. PMID:23760508

Reactive oxygen species inactivate neuronal nicotinic acetylcholine receptors through a highly conserved cysteine near the intracellular mouth of the channel: implications for diseases that involve oxidative stress

PubMed Central

Krishnaswamy, Arjun; Cooper, Ellis

2012-01-01

Abstract An intriguing feature of several nicotinic acetylcholine receptors (nAChRs) on neurons is that their subunits contain a highly conserved cysteine residue located near the intracellular mouth of the receptor pore. The work summarized in this review indicates that α3β4-containing and α4β2-containing neuronal nAChRs, and possibly other subtypes, are inactivated by elevations in intracellular reactive oxygen species (ROS). This review discusses a model for the molecular mechanisms that underlie this inactivation. In addition, we explore the implications of this mechanism in the context of complications that arise from diabetes. We review the evidence that diabetes elevates cytosolic ROS in sympathetic neurons and inactivates postsynaptic α3β4-containing nAChRs shortly after the onset of diabetes, leading to a depression of synaptic transmission in sympathetic ganglia, an impairment of sympathetic reflexes. These effects of ROS on nAChR function are due to the highly conserved Cys residues in the receptors: replacing the cysteine residues in α3 allow ganglionic transmission and sympathetic reflexes to function normally in diabetes. This example from diabetes suggests that other diseases involving oxidative stress, such as Parkinson's disease, could lead to the inactivation of nAChRs on neurons and disrupt cholinergic nicotinic signalling. PMID:21969449

The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor.

PubMed Central

Parrish, William; Eilers, Markus; Ying, Weiwen; Konopka, James B

2002-01-01

The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors. PMID:11861550

Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Auperin,T.; Bolduc, G.; Baron, M.

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} ofmore » the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.« less

Automated hierarchical classification of protein domain subfamilies based on functionally-divergent residue signatures

PubMed Central

2012-01-01

Background The NCBI Conserved Domain Database (CDD) consists of a collection of multiple sequence alignments of protein domains that are at various stages of being manually curated into evolutionary hierarchies based on conserved and divergent sequence and structural features. These domain models are annotated to provide insights into the relationships between sequence, structure and function via web-based BLAST searches. Results Here we automate the generation of conserved domain (CD) hierarchies using a combination of heuristic and Markov chain Monte Carlo (MCMC) sampling procedures and starting from a (typically very large) multiple sequence alignment. This procedure relies on statistical criteria to define each hierarchy based on the conserved and divergent sequence patterns associated with protein functional-specialization. At the same time this facilitates the sequence and structural annotation of residues that are functionally important. These statistical criteria also provide a means to objectively assess the quality of CD hierarchies, a non-trivial task considering that the protein subgroups are often very distantly related—a situation in which standard phylogenetic methods can be unreliable. Our aim here is to automatically generate (typically sub-optimal) hierarchies that, based on statistical criteria and visual comparisons, are comparable to manually curated hierarchies; this serves as the first step toward the ultimate goal of obtaining optimal hierarchical classifications. A plot of runtimes for the most time-intensive (non-parallelizable) part of the algorithm indicates a nearly linear time complexity so that, even for the extremely large Rossmann fold protein class, results were obtained in about a day. Conclusions This approach automates the rapid creation of protein domain hierarchies and thus will eliminate one of the most time consuming aspects of conserved domain database curation. At the same time, it also facilitates protein domain annotation by identifying those pattern residues that most distinguish each protein domain subgroup from other related subgroups. PMID:22726767

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation.

PubMed

Zattas, Dimitrios; Berk, Jason M; Kreft, Stefan G; Hochstrasser, Mark

2016-06-03

Specific proteins are modified by ubiquitin at the endoplasmic reticulum (ER) and are degraded by the proteasome, a process referred to as ER-associated protein degradation. In Saccharomyces cerevisiae, two principal ER-associated protein degradation ubiquitin ligases (E3s) reside in the ER membrane, Doa10 and Hrd1. The membrane-embedded Doa10 functions in the degradation of substrates in the ER membrane, nuclear envelope, cytoplasm, and nucleoplasm. How most E3 ligases, including Doa10, recognize their protein substrates remains poorly understood. Here we describe a previously unappreciated but highly conserved C-terminal element (CTE) in Doa10; this cytosolically disposed 16-residue motif follows the final transmembrane helix. A conserved CTE asparagine residue is required for ubiquitylation and degradation of a subset of Doa10 substrates. Such selectivity suggests that the Doa10 CTE is involved in substrate discrimination and not general ligase function. Functional conservation of the CTE was investigated in the human ortholog of Doa10, MARCH6 (TEB4), by analyzing MARCH6 autoregulation of its own degradation. Mutation of the conserved Asn residue (N890A) in the MARCH6 CTE stabilized the normally short lived enzyme to the same degree as a catalytically inactivating mutation (C9A). We also report the localization of endogenous MARCH6 to the ER using epitope tagging of the genomic MARCH6 locus by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. These localization and CTE analyses support the inference that MARCH6 and Doa10 are functionally similar. Moreover, our results with the yeast enzyme suggest that the CTE is involved in the recognition and/or ubiquitylation of specific protein substrates. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The solution structure of omega-Aga-IVB, a P-type calcium channel antagonist from venom of the funnel web spider, Agelenopsis aperta.

PubMed

Reily, M D; Thanabal, V; Adams, M E

1995-02-01

The 48 amino acid peptides omega-Aga-IVA and omega-Aga-IVB are the first agents known to specifically block P-type calcium channels in mammalian brain, thus complementing the existing suite of pharmacological tools used for characterizing calcium channels. These peptides provide a new set of probes for studies aimed at elucidating the structural basis underlying the subtype specificity of calcium channel antagonists. We used 288 NMR-derived constraints in a protocol combining distance geometry and molecular dynamics employing the program DGII, followed by energy minimization with Discover to derive the three-dimensional structure of omega-Aga-IVB. The toxin consists of a well-defined core region, comprising seven solvent-shielded residues and a well-defined triple-stranded beta-sheet. Four loop regions have average backbone rms deviations between 0.38 and 1.31 A, two of which are well-defined type-II beta-turns. Other structural features include disordered C- and N-termini and several conserved basic amino acids that are clustered on one face of the molecule. The reported structure suggests a possible surface for interaction with the channel. This surface contains amino acids that are identical to those of another known P-type calcium channel antagonist, omega-Aga-IVA, and is rich in basic residues that may have a role in binding to the anionic sites in the extracellular regions of the calcium channel.

Energy conservation policy evaluation. Study module IA. Volume II. Technical appendix. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bergstresser, K.; Berney, R.E.; Carter, L.F.

1978-01-01

This volume contains detailed technical analyses of the 19 energy conservation measures which were discussed as regional policy options in the report proper (PB-274 337). For each measure there is a description of how the measure works to reduce consumption; an assessment of potential savings, costs, and returns to adopters; probable 'normal' adoption rates under present policies; and potential adoption rates if some additional conservation policy options are implemented. Report includes chapters on environmental residuals from energy end uses, and policy preferences of households and energy marketers.

NMR structure of the viral peptide linked to the genome (VPg) of poliovirus

PubMed Central

Schein, Catherine H.; Oezguen, Numan; Volk, David E.; Garimella, Ravindranath; Paul, Aniko; Braun, Werner

2006-01-01

VPgs are essential for replication of picornaviruses, which cause diseases such as poliomyelitis, foot and mouth disease, and the common cold. VPg in infected cells is covalently linked to the 5' end of the viral RNA, or, in a uridylylated form, free in the cytoplasm. We show here the first solution structure for a picornaviral VPg, that of the 22-residue peptide from poliovirus serotype 1. VPg in buffer is inherently flexible, but a single conformer was obtained by adding trimethylamine N-oxide (TMAO). TMAO had only minor effects on the TOCSY spectrum. However, it increased the amount of structured peptide, as indicated by more peaks in the NOESY spectrum and an up to 300% increase in the ratio of normalized NOE crosspeak intensities to that in buffer. The data for VPg in TMAO yielded a well defined structure bundle with 0.6Å RMSD (vs. 6.6 Å in buffer alone), with 10–30 unambiguous constraints per residue. The structure consists of a large loop region from residues 1–14, from which the reactive tyrosinate projects outward, and a C-terminal helix from residues 18–21 that aligns the sidechains of conserved residues on one face. The structure has a stable docking position at an area on the poliovirus polymerase crystal structure identified as a VPg binding site by mutagenesis studies. Further, UTP and ATP dock in a base-specific manner to the reactive face of VPg, held in place by residues conserved in all picornavirus VPgs. PMID:16540201

Effects of cysteine introduction into three homologous cytochromes C.

PubMed

Kobayashi, Yoshiko; Sonoyama, Takafumi; Takeda, Taku; Sambongi, Yoshihiro

2009-05-01

A cysteine residue was systematically introduced into three homologous cytochromes c from Hydrogenobacter thermophilus, Hydrogenophilus thermoluteolus, and Pseudomonas aeruginosa at a conserved position. The H. thermoluteolus variant showed the most decreased thermal stability as compared with the wild type, which might have been due in part to crosslinked polymer formation. The effects of cysteine introduction differed even at the conserved position in these homologous proteins.

Combining Landsat-8 and WorldView-3 data to assess crop residue cover

USDA-ARS?s Scientific Manuscript database

Crop residues on the soil surface contribute to soil quality and form the first line defense against the erosive forces of water and wind. Quantifying crop residue cover on the soil surface after crops are planted is crucial for monitoring soil tillage intensity and assessing the extent of conserva...

Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface.

PubMed

Machius, Mischa; Declerck, Nathalie; Huber, Robert; Wiegand, Georg

2003-03-28

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.

Effect of crop residue harvest on long-term crop yield, soil erosion, and carbon balance: tradeoffs for a sustainable bioenergy feedstock

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gregg, Jay S.; Izaurralde, Roberto C.

2010-08-26

Agricultural residues are a potential feedstock for bioenergy production, if residue harvest can be done sustainably. The relationship between crop residue harvest, soil erosion, crop yield and carbon balance was modeled with the Erosion Productivity Impact Calculator/ Environment Policy Integrated Climate (EPIC) using a factorial design. Four crop rotations (winter wheat [Triticum aestivum (L.)] – sunflower [Helianthus annuus]; spring wheat [Triticum aestivum (L.)] – canola [Brassica napus]; corn [Zea mays L.] – soybean [Glycine max (L.) Merr.]; and cotton [Gossypium hirsutum] – peanut [Arachis hypogaea]) were simulated at four US locations each, under different topographies (0-10% slope), and management practicesmore » [crop residue removal rates (0-75%), conservation practices (no till, contour cropping, strip cropping, terracing)].« less

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels

PubMed Central

Shang, Lijun

2007-01-01

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K+ channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the “helix-bundle crossing”. However, in the inwardly rectifying (Kir) potassium channel family, the role of this “hinge” residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper “hinge” residues are in close contact with the base of the pore α-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the “lower” gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue. PMID:17657484

Autoacetylation of the Ralstonia solanacearum effector PopP2 targets a lysine residue essential for RRS1-R-mediated immunity in Arabidopsis.

PubMed

Tasset, Céline; Bernoux, Maud; Jauneau, Alain; Pouzet, Cécile; Brière, Christian; Kieffer-Jacquinod, Sylvie; Rivas, Susana; Marco, Yves; Deslandes, Laurent

2010-11-18

Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as "guards". The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity.

Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3 Cytochrome Oxidase.

PubMed

Funatogawa, Chie; Li, Yang; Chen, Ying; McDonald, William; Szundi, Istvan; Fee, James A; Stout, C David; Einarsdóttir, Ólöf

2017-01-10

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba 3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O 2 , and NO binding were investigated. O 2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O-O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a 3 during O-O bond cleavage in V236A, followed by faster re-reduction of heme b by Cu A . This interpretation is supported by classical molecular dynamics simulations of diffusion of O 2 to the active site in V236A that indicated a larger distance between the two hemes compared to that in the wild type and increased contact of heme a 3 with water and weakened interactions with residues R444 and R445. As the size of the mutant side chain increased and protruded more into the ligand cavity, the rates of ligand binding decreased correspondingly. These results demonstrate the importance of V236 in facilitating access of ligands to the active site in T. thermophilus ba 3 .

On the Role of Aggregation Prone Regions in Protein Evolution, Stability, and Enzymatic Catalysis: Insights from Diverse Analyses

PubMed Central

Buck, Patrick M.; Kumar, Sandeep; Singh, Satish K.

2013-01-01

The various roles that aggregation prone regions (APRs) are capable of playing in proteins are investigated here via comprehensive analyses of multiple non-redundant datasets containing randomly generated amino acid sequences, monomeric proteins, intrinsically disordered proteins (IDPs) and catalytic residues. Results from this study indicate that the aggregation propensities of monomeric protein sequences have been minimized compared to random sequences with uniform and natural amino acid compositions, as observed by a lower average aggregation propensity and fewer APRs that are shorter in length and more often punctuated by gate-keeper residues. However, evidence for evolutionary selective pressure to disrupt these sequence regions among homologous proteins is inconsistent. APRs are less conserved than average sequence identity among closely related homologues (≥80% sequence identity with a parent) but APRs are more conserved than average sequence identity among homologues that have at least 50% sequence identity with a parent. Structural analyses of APRs indicate that APRs are three times more likely to contain ordered versus disordered residues and that APRs frequently contribute more towards stabilizing proteins than equal length segments from the same protein. Catalytic residues and APRs were also found to be in structural contact significantly more often than expected by random chance. Our findings suggest that proteins have evolved by optimizing their risk of aggregation for cellular environments by both minimizing aggregation prone regions and by conserving those that are important for folding and function. In many cases, these sequence optimizations are insufficient to develop recombinant proteins into commercial products. Rational design strategies aimed at improving protein solubility for biotechnological purposes should carefully evaluate the contributions made by candidate APRs, targeted for disruption, towards protein structure and activity. PMID:24146608

Effect of Applied Stress and Temperature on Residual Stresses Induced by Peening Surface Treatments in Alloy 600

NASA Astrophysics Data System (ADS)

Telang, A.; Gnäupel-Herold, T.; Gill, A.; Vasudevan, V. K.

2018-04-01

In this study, the effects of applied tensile stress and temperature on laser shock peening (LSP) and cavitation shotless peening (CSP)-induced compressive residual stresses were investigated using neutron and x-ray diffraction. Residual stresses on the surface, measured in situ, were lower than the applied stress in LSP- and CSP-treated Alloy 600 samples (2 mm thick). The residual stress averaged over the volume was similar to the applied stress. Compressive residual stresses on the surface and balancing tensile stresses in the interior relax differently due to hardening induced by LSP. Ex situ residual stress measurements, using XRD, show that residual stresses relaxed as the applied stress exceeded the yield strength of the LSP- and CSP-treated Alloy 600. Compressive residual stresses induced by CSP and LSP decreased by 15-25% in magnitude, respectively, on exposure to 250-450 °C for more than 500 h with 10-11% of relaxation occurring in the first few hours. Further, 80% of the compressive residual stresses induced by LSP and CSP treatments in Alloy 600 were retained even after long-term aging at 350 °C for 2400 h.

Net global warming potential and greenhouse gas intensity of conventional and conservation agriculture system in rainfed semi arid tropics of India

NASA Astrophysics Data System (ADS)

Pratibha, G.; Srinivas, I.; Rao, K. V.; Shanker, Arun K.; Raju, B. M. K.; Choudhary, Deepak K.; Srinivas Rao, K.; Srinivasarao, Ch.; Maheswari, M.

2016-11-01

Agriculture has been considered as one of the contributors to greenhouse gas (GHG) emissions and it continues to increase with increase in crop production. Hence development of sustainable agro techniques with maximum crop production, and low global warming potential is need of the hour. Quantifying net global warming potential (NGWP) and greenhouse gas intensity (GHGI) of an agricultural activity is a method to assess the mitigation potential of the activity. But there is dearth of information on NGWP of conservation agriculture under rainfed conditions. Hence in this study two methods such as crop based (NGWPcrop) and soil based (NGWPsoil) were estimated from the data of the experiment initiated in 2009 in rainfed semiarid regions of Hyderabad, India with different tillage practices like conventional tillage (CT), reduced tillage (RT), zero tillage (ZT) and residue retention levels by harvesting at different heights which includes 0, 10 and 30 cm anchored residue in pigeonpea-castor systems. The results of the study revealed that under rainfed conditions CT recorded 24% higher yields over ZT, but CT and RT were on par with each other. However, the yield gap between the tillage treatments is narrowing down over 5 years of study. ZT and RT recorded 26 and 11% lower indirect GHG emissions (emissions from farm operations and input use) over CT, respectively. The percent contribution of CO2 eq. N2O emission is higher to total GHG emissions in both the crops. Both NGWPcrop, NGWPsoil, GHGIcrop, and GHGIsoil based were influenced by tillage and residue treatments. Further, castor grown on pigeonpea residue recorded 20% higher GHG emissions over pigeonpea grown on castor residues. The fuel consumption in ZT was reduced by 58% and 81% as compared to CT in pigeonpea and castor, respectively. Lower NGWP and GHGI based on crop and soil was observed with increase in crop residues and decrease in tillage intensity in both the crops. The results of the study indicate that, there is scope to reduce the NGWP emissions by reducing one tillage operation as in RT and increase in crop residue by harvesting at 10 and 30 cm height with minimal impact on the crop yields. However, the trade-off between higher yield and soil health versus GHG emissions should be considered while promoting conservation agriculture. The NGWPcrop estimation method indicated considerable benefits of residues to the soil and higher potential of GHG mitigation than by the NGWPsoil method and may overestimate the potential of GHG mitigation in agriculture system.

Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.

Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 andmore » A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential contribution to product release.« less

Structural studies and molecular dynamics simulations suggest a processive mechanism of exolytic lytic transglycosylase from Campylobacter jejuni.

PubMed

Vijayaraghavan, Jagamya; Kumar, Vijay; Krishnan, Nikhil P; Kaufhold, Ross T; Zeng, Ximin; Lin, Jun; van den Akker, Focco

2018-01-01

The bacterial soluble lytic transglycosylase (LT) breaks down the peptidoglycan (PG) layer during processes such as cell division. We present here crystal structures of the soluble LT Cj0843 from Campylobacter jejuni with and without bulgecin A inhibitor in the active site. Cj0843 has a doughnut shape similar but not identical to that of E. coli SLT70. The C-terminal catalytic domain is preceded by an L-domain, a large helical U-domain, a flexible linker, and a small N-terminal NU-domain. The flexible linker allows the NU-domain to reach over and complete the circular shape, using residues conserved in the Epsilonproteobacteria LT family. The inner surface of the Cj0843 doughnut is mostly positively charged including a pocket that has 8 Arg/Lys residues. Molecular dynamics simulations with PG strands revealed a potential functional role for this pocket in anchoring the negatively charged terminal tetrapeptide of the PG during several steps in the reaction including homing and aligning the PG strand for exolytic cleavage, and subsequent ratcheting of the PG strand to enhance processivity in degrading PG strands.

Kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab) coat protein and its mutants with ssDNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Priyadarshini, C.G. Poornima; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.i

Gemini viral assembly and transport of viral DNA into nucleus for replication, essentially involve DNA-coat protein interactions. The kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali recombinant coat protein (rCP) with DNA was studied by electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR). The rCP interacted with ssDNA with a K{sub A}, of 2.6 +- 0.29 x 10{sup 8} M{sup -1} in a sequence non-specific manner. The CP has a conserved C2H2 type zinc finger motif composed of residues C68, C72, H81 and H85. Mutation of these residues to alanine resulted in reduced binding to DNA probes.more » The H85A mutant rCP showed the least binding with approximately 756 fold loss in the association rate and a three order magnitude decrease in the binding affinity as compared to rCP. The CP-DNA interactions via the zinc finger motif could play a crucial role in virus assembly and in nuclear transport.« less

Evidence that electrostatic interactions between vesicle-associated membrane protein 2 and acidic phospholipids may modulate the fusion of transport vesicles with the plasma membrane.

PubMed

Williams, Dumaine; Vicôgne, Jérome; Zaitseva, Irina; McLaughlin, Stuart; Pessin, Jeffrey E

2009-12-01

The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in beta cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.

The RNA-binding protein Hfq is important for ribosome biogenesis and affects translation fidelity.

PubMed

Andrade, José M; Dos Santos, Ricardo F; Chelysheva, Irina; Ignatova, Zoya; Arraiano, Cecília M

2018-06-01

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA-mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA Hfq assists ribosome assembly and associates with pre-30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq-mediated regulation of ribosomes is independent of its function as sRNA-regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm-like protein Hfq beyond its function in small RNA-mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation. © 2018 The Authors.

Insights into the functional role of protonation states in the HIV-1 protease-BEA369 complex: molecular dynamics simulations and free energy calculations.

PubMed

Chen, Jianzhong; Yang, Maoyou; Hu, Guodong; Shi, Shuhua; Yi, Changhong; Zhang, Qinggang

2009-10-01

The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method combined with molecular dynamics (MD) simulations were used to investigate the functional role of protonation in human immunodeficiency virus type 1 (HIV-1) protease complexed with the inhibitor BEA369. Our results demonstrate that protonation of two aspartic acids (Asp25/Asp25') has a strong influence on the dynamics behavior of the complex, the binding free energy of BEA369, and inhibitor-residue interactions. Relative binding free energies calculated using the MM-PBSA method show that protonation of Asp25 results in the strongest binding of BEA369 to HIV-1 protease. Inhibitor-residue interactions computed by the theory of free energy decomposition also indicate that protonation of Asp25 has the most favorable effect on binding of BEA369. In addition, hydrogen-bond analysis based on the trajectories of the MD simulations shows that protonation of Asp25 strongly influences the water-mediated link of a conserved water molecule, Wat301. We expect that the results of this study will contribute significantly to binding calculations for BEA369, and to the design of high affinity inhibitors.

The Efficiency of Dentin Sialoprotein-Phosphophoryn Processing Is Affected by Mutations Both Flanking and Distant from the Cleavage Site*

PubMed Central

Yang, Robert T.; Lim, Glendale L.; Dong, Zhihong; Lee, Arthur M.; Yee, Colin T.; Fuller, Robert S.; Ritchie, Helena H.

2013-01-01

Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G447↓D448 cleavage site in DSP-PP240 had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P4 to P4′ blocked, impaired, or enhanced DSP-PP240 cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP240 had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP240 significantly modified the amount of PP240 product generated from DSP-PP240 precursor protein cleavage suggests that such mutation may affect the mineralization process. PMID:23297400

The efficiency of dentin sialoprotein-phosphophoryn processing is affected by mutations both flanking and distant from the cleavage site.

PubMed

Yang, Robert T; Lim, Glendale L; Dong, Zhihong; Lee, Arthur M; Yee, Colin T; Fuller, Robert S; Ritchie, Helena H

2013-02-22

Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G(447)↓D(448) cleavage site in DSP-PP(240) had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P(4) to P(4)' blocked, impaired, or enhanced DSP-PP(240) cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP(240) had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP(240) significantly modified the amount of PP(240) product generated from DSP-PP(240) precursor protein cleavage suggests that such mutation may affect the mineralization process.

Structure of genes for dermaseptins B, antimicrobial peptides from frog skin. Exon 1-encoded prepropeptide is conserved in genes for peptides of highly different structures and activities.

PubMed

Vouille, V; Amiche, M; Nicolas, P

1997-09-01

We cloned the genes of two members of the dermaseptin family, broad-spectrum antimicrobial peptides isolated from the skin of the arboreal frog Phyllomedusa bicolor. The dermaseptin gene Drg2 has a 2-exon coding structure interrupted by a small 137-bp intron, wherein exon 1 encoded a 22-residue hydrophobic signal peptide and the first three amino acids of the acidic propiece; exon 2 contained the 18 additional acidic residues of the propiece plus a typical prohormone processing signal Lys-Arg and a 32-residue dermaseptin progenitor sequence. The dermaseptin genes Drg2 and Drg1g2 have conserved sequences at both untranslated ends and in the first and second coding exons. In contrast, Drg1g2 comprises a third coding exon for a short version of the acidic propiece and a second dermaseptin progenitor sequence. Structural conservation between the two genes suggests that Drg1g2 arose recently from an ancestral Drg2-like gene through amplification of part of the second coding exon and 3'-untranslated region. Analysis of the cDNAs coding precursors for several frog skin peptides of highly different structures and activities demonstrates that the signal peptides and part of the acidic propieces are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The organization of the genes that belong to this family, with the signal peptide and the progenitor sequence on separate exons, permits strikingly different peptides to be directed into the secretory pathway. The recruitment of such a homologous 'secretory' exon by otherwise non-homologous genes may have been an early event in the evolution of amphibian.

A comparative, prospective, and randomized study of two conservative treatment protocols for first-episode lateral ankle ligament injuries.

PubMed

Prado, Marcelo Pires; Mendes, Alberto Abussamra Moreira; Amodio, Daniel Tasseto; Camanho, Gilberto Luis; Smyth, Niall A; Fernandes, Tulio Diniz

2014-03-01

The objective of this study was to investigate functional results, the amount of time that patients missed from regular working activities, and the incidence of residual mechanical ankle instability following conservative treatment of a first episode of severe lateral ankle ligament sprain (with articular instability). This prospective and randomized study included 186 patients with severe lateral ankle ligament injuries, who were randomly assigned into 2 conservative treatment groups. In group A, participants were treated with a walking boot with weight-bearing allowed, pain management, ice, and elevation with restricted joint mobilization for 3 weeks. In group B, patients were treated with a functional brace for 3 weeks. After this period, patients from both groups were placed in a short, functional brace for an additional 3 weeks, during which they also started a rehabilitation program. No statistically significant difference was found in pain intensity score between the 2 groups; however, functional evaluations based on the AOFAS ankle and hindfoot score system showed a statistically significant improvement in the group treated with the functional brace. In addition, the average recovery period necessary for patients of group B to resume their duties was shorter than that for patients in group A. No significant difference was detected in residual mechanical ankle instability between the 2 groups. Patients with severe lateral ankle ligament lesions treated with a functional brace were shown to exhibit somewhat better results than those treated with a walking boot, and both methods presented a very low incidence of residual chronic instability. We found adequate conservative treatment was sufficient to reestablish ankle stability and that functional treatment had a marginally better clinical short-term outcome with a shorter average recovery period. Level I, prospective randomized study.

An evolutionarily conserved motif in the TAB1 C-terminal region is necessary for interaction with and activation of TAK1 MAPKKK.

PubMed

Ono, K; Ohtomo, T; Sato, S; Sugamata, Y; Suzuki, M; Hisamoto, N; Ninomiya-Tsuji, J; Tsuchiya, M; Matsumoto, K

2001-06-29

TAK1, a member of the MAPKKK family, is involved in the intracellular signaling pathways mediated by transforming growth factor beta, interleukin 1, and Wnt. TAK1 kinase activity is specifically activated by the TAK1-binding protein TAB1. The C-terminal 68-amino acid sequence of TAB1 (TAB1-C68) is sufficient for TAK1 interaction and activation. Analysis of various truncated versions of TAB1-C68 defined a C-terminal 30-amino acid sequence (TAB1-C30) necessary for TAK1 binding and activation. NMR studies revealed that the TAB1-C30 region has a unique alpha-helical structure. We identified a conserved sequence motif, PYVDXA/TXF, in the C-terminal domain of mammalian TAB1, Xenopus TAB1, and its Caenorhabditis elegans homolog TAP-1, suggesting that this motif constitutes a specific TAK1 docking site. Alanine substitution mutagenesis showed that TAB1 Phe-484, located in the conserved motif, is crucial for TAK1 binding and activation. The C. elegans homolog of TAB1, TAP-1, was able to interact with and activate the C. elegans homolog of TAK1, MOM-4. However, the site in TAP-1 corresponding to Phe-484 of TAB1 is an alanine residue (Ala-364), and changing this residue to Phe abrogates the ability of TAP-1 to interact with and activate MOM-4. These results suggest that the Phe or Ala residue within the conserved motif of the TAB1-related proteins is important for interaction with and activation of specific TAK1 MAPKKK family members in vivo.

Structural Basis of Conserved Cysteine in the Fibroblast Growth Factor Family: Evidence for a Vestigial Half-Cystine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jihun; Blaber, Michael; FSU)

2010-11-09

The 22 members of the mouse/human fibroblast growth factor (FGF) family of proteins contain a conserved cysteine residue at position 83 (numbering scheme of the 140-residue form of FGF-1). Sequence and structure information suggests that this position is a free cysteine in 16 members and participates as a half-cystine in at least 3 (and perhaps as many as 6) other members. While a structural role as a half-cystine provides a stability basis for possible selective pressure, it is less clear why this residue is conserved as a free cysteine (although free buried thiols can limit protein functional half-life). To probemore » the structural role of the free cysteine at position 83 in FGF-1, we constructed Ala, Ser, Thr, Val, and Ile mutations and determined their effects on structure and stability. These results show that position 83 in FGF-1 is thermodynamically optimized to accept a free cysteine. A second cysteine mutation was introduced into wild-type FGF-1 at adjacent position Ala66, which is known to participate as a half-cystine with position 83 in FGF-8, FGF-19, and FGF-23. Results show that, unlike position 83, a free cysteine at position 66 destabilizes FGF-1; however, upon oxidation, a near-optimal disulfide bond is formed between Cys66 and Cys83, resulting in {approx} 14 kJ/mol of increased thermostability. Thus, while the conserved free cysteine at position 83 in the majority of the FGF proteins may have a principal role in limiting functional half-life, evidence suggests that it is a vestigial half-cystine.« less

Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

NASA Technical Reports Server (NTRS)

Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

1995-01-01

A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

CHARACTERIZING TRANSFER OF SURFACE RESIDUES TO SKIN USING A VIDEO-FLUORESCENT IMAGING TECHNIQUE

EPA Science Inventory

Surface-to-skin transfer of contaminants is a complex process. For children's residential exposure, transfer of chemicals from contaminated surfaces such as floors and furniture is potentially significant. Once on the skin, residues and contaminated particles can be transferred b...

Trend-surface analysis of the structure of the Ste. Genevieve limestone in the Effingham, Illinois, area

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stevenson, D.

1970-01-01

Recent studies of oil accumulations in the Ste. Genevieve Limestone Formation in Illinois demonstrate the usefulness of fitting third-order trend surfaces to structural data and analyzing the residuals calculated by subtracting the trend surface from the structure surface. Known oil pools are located in areas having positive residual values. This type of investigation, supplemented by conventional structural and stratigraphic studies, was performed on a 9-township (approx. 324 sq miles) area in Effingham and Shelby counties, Illinois. The known oil pools in the oolite and sandstone lenses of the Ste. Genevieve Formation lie within positive residuals resulting from the difference betweenmore » a third-order trend surface and the structural surface on top of the Ste. Genevieve. A composite map outlining areas where present anticlinal noses, sandstone lenses, and positive residuals lie in close proximity to each other is included in this report to indicate places where future exploration for Ste. Genevieve oil would have the greatest chance for success.« less

Free Energy Landscape - Settlements of Key Residues.

NASA Astrophysics Data System (ADS)

Aroutiounian, Svetlana

2007-03-01

FEL perspective in studies of protein folding transitions reflects notion that since there are ˜10^N conformations to scan in search of lowest free energy state, random search is beyond biological timescale. Protein folding must follow certain fel pathways and folding kinetics of evolutionary selected proteins dominates kinetic traps. Good model for functional robustness of natural proteins - coarse-grained model protein is not very accurate but affords bringing simulations closer to biological realm; Go-like potential secures the fel funnel shape; biochemical contacts signify the funnel bottleneck. Boltzmann-weighted ensemble of protein conformations and histogram method are used to obtain from MC sampling of protein conformational space the approximate probability distribution. The fel is F(rmsd) = -1/βLn[Hist(rmsd)], β=kBT and rmsd is root-mean-square-deviation from native conformation. The sperm whale myoglobin has rich dynamic behavior, is small and large - on computational scale, has a symmetry in architecture and unusual sextet of residue pairs. Main idea: there is a mathematical relation between protein fel and a key residues set providing stability to folding transition. Is the set evolutionary conserved also for functional reasons? Hypothesis: primary sequence determines the key residues positions conserved as stabilizers and the fel is the battlefield for the folding stability. Preliminary results: primary sequence - not the architecture, is the rule settler, indeed.

Identification of a residue in helix 2 of rice plasma membrane intrinsic proteins that influences water permeability.

PubMed

Zhang, Minhua; Lü, Shouqin; Li, Guowei; Mao, Zhilei; Yu, Xin; Sun, Weining; Tang, Zhangcheng; Long, Mian; Su, Weiai

2010-12-31

Molecular selection, ion exclusion, and water permeation are well known regulatory mechanisms in aquaporin. Water permeability was found to be diverse in different subgroups of plasma membrane intrinsic proteins (PIPs), even though the residues surrounding the water holes remained the same across the subgroups. Upon homology modeling and structural comparison, a conserved Ala/Ile(Val) residue difference was identified in helix 2 that affected the conformation of the NPA region and consequently influenced the water permeability. The residue difference was found to be conservative within the two subgroups of PIPs in rice as well as in other plants. Functional tests further confirmed the prediction via site-directed mutagenesis where replacement of Ala(103) or Ala(102) in respective OsPIP1;1 or OsPIP1;3 with Val yielded 7.0- and 2.2-fold increases in water transportation, and substitution of Ile(98) or Val(95) in respective OsPIP2;3 or OsPIP2;7 with Ala resulted in 73 or 52% reduction of water transportation. Based on structural analyses and molecular dynamics simulations, we proposed that the difference in water permeability was attributed to the orientation variations of helix 2 that modified water-water and water-protein interactions.

Voltage Sensing in Membranes: From Macroscopic Currents to Molecular Motions.

PubMed

Freites, J Alfredo; Tobias, Douglas J

2015-06-01

Voltage-sensing domains (VSDs) are integral membrane protein units that sense changes in membrane electric potential, and through the resulting conformational changes, regulate a specific function. VSDs confer voltage-sensitivity to a large superfamily of membrane proteins that includes voltage-gated Na[Formula: see text], K[Formula: see text], Ca[Formula: see text] ,and H[Formula: see text] selective channels, hyperpolarization-activated cyclic nucleotide-gated channels, and voltage-sensing phosphatases. VSDs consist of four transmembrane segments (termed S1 through S4). Their most salient structural feature is the highly conserved positions for charged residues in their sequences. S4 exhibits at least three conserved triplet repeats composed of one basic residue (mostly arginine) followed by two hydrophobic residues. These S4 basic side chains participate in a state-dependent internal salt-bridge network with at least four acidic residues in S1-S3. The signature of voltage-dependent activation in electrophysiology experiments is a transient current (termed gating or sensing current) upon a change in applied membrane potential as the basic side chains in S4 move across the membrane electric field. Thus, the unique structural features of the VSD architecture allow for competing requirements: maintaining a series of stable transmembrane conformations, while allowing charge motion, as briefly reviewed here.

Functional Divergence of FimX in PilZ Binding and Type IV Pilus Regulation

PubMed Central

Qi, Yaning; Xu, Linghui; Dong, Xueming; Yau, Yin Hoe; Ho, Chun Loong; Koh, Siew Lee; Shochat, Susana Geifman; Chou, Shan-Ho; Tang, Kai

2012-01-01

Type IV pili (T4P) are polar surface structures that play important roles in bacterial motility, biofilm formation, and pathogenicity. The protein FimX and its orthologs are known to mediate T4P formation in the human pathogen Pseudomonas aeruginosa and some other bacterial species. It was reported recently that FimXXAC2398 from Xanthomonas axonopodis pv. citri interacts with PilZXAC1133 directly through the nonenzymatic EAL domain of FimXXAC2398. Here we present experimental data to reveal that the strong interaction between FimXXAC2398 and PilZXAC1133 is not conserved in P. aeruginosa and likely other Pseudomonas species. In vitro and in vivo binding experiments showed that the interaction between FimX and PilZ in P. aeruginosa is below the measurable limit. Surface plasmon resonance assays further confirmed that the interaction between the P. aeruginosa proteins is at least more than 3 orders of magnitude weaker than that between the X. axonopodis pv. citri pair. The N-terminal lobe region of FimXXAC2398 was identified as the binding surface for PilZXAC1133 by amide hydrogen-deuterium exchange and site-directed mutagenesis studies. Lack of several key residues in the N-terminal lobe region of the EAL domain of FimX is likely to account for the greatly reduced binding affinity between FimX and PilZ in P. aeruginosa. All together, the results suggest that the interaction between PilZ and FimX in Xanthomonas species is not conserved in P. aeruginosa due to the evolutionary divergence among the FimX orthologs. The precise roles of FimX and PilZ in bacterial motility and T4P biogenesis are likely to vary among bacterial species. PMID:22942245

High-level systemic expression of conserved influenza epitope in plants on the surface of rod-shaped chimeric particles.

PubMed

Petukhova, Natalia V; Gasanova, Tatiana V; Ivanov, Peter A; Atabekov, Joseph G

2014-04-21

Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV) genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP) gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV) did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP) described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.