An Interdomain Interaction of the Androgen Receptor Is Required for Its Aggregation and Toxicity in Spinal and Bulbar Muscular Atrophy*

PubMed Central

Orr, Christopher R.; Montie, Heather L.; Liu, Yuhong; Bolzoni, Elena; Jenkins, Shannon C.; Wilson, Elizabeth M.; Joseph, James D.; McDonnell, Donald P.; Merry, Diane E.

2010-01-01

Polyglutamine expansion within the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA) and is associated with misfolded and aggregated species of the mutant AR. We showed previously that nuclear localization of the mutant AR was necessary but not sufficient for SBMA. Here we show that an interdomain interaction of the AR that is central to its function within the nucleus is required for AR aggregation and toxicity. Ligands that prevent the interaction between the amino-terminal FXXLF motif and carboxyl-terminal AF-2 domain (N/C interaction) prevented toxicity and AR aggregation in an SBMA cell model and rescued primary SBMA motor neurons from 5α-dihydrotestosterone-induced toxicity. Moreover, genetic mutation of the FXXLF motif prevented AR aggregation and 5α-dihydrotestosterone toxicity. Finally, selective androgen receptor modulators, which prevent the N/C interaction, ameliorated AR aggregation and toxicity while maintaining AR function, highlighting a novel therapeutic strategy to prevent the SBMA phenotype while retaining AR transcriptional function. PMID:20826791

Experimentally biased model structure of the Hsc70/auxilin complex: Substrate transfer and interdomain structural change

PubMed Central

Gruschus, James M.; Greene, Lois E.; Eisenberg, Evan; Ferretti, James A.

2004-01-01

A model structure of the Hsc70/auxilin complex has been constructed to gain insight into interprotein substrate transfer and ATP hydrolysis induced conformational changes in the multidomain Hsc70 structure. The Hsc70/auxilin system, which is a member of the Hsp70/Hsp40 chaperone system family, uncoats clathrin-coated vesicles in an ATP hydrolysis-driven process. Incorporating previous results from NMR and mutant binding studies, the auxilin J-domain was docked into the Hsc70 ATPase domain lower cleft using rigid backbone/flexible side chain molecular dynamics, and the Hsc70 substrate binding domain was docked by a similar procedure. For comparison, J-domain and substrate binding domain docking sites were obtained by the rigid-body docking programs DOT and ZDOCK, filtered and ranked by the program ClusPro, and relaxed using the same rigid backbone/flexible side chain dynamics. The substrate binding domain sites were assessed in terms of conserved surface complementarity and feasibility in the context of substrate transfer, both for auxilin and another Hsp40 protein, Hsc20. This assessment favors placement of the substrate binding domain near D152 on the ATPase domain surface adjacent to the J-domain invariant HPD segment, with the Hsc70 interdomain linker in the lower cleft. Examining Hsc70 interdomain energetics, we propose that long-range electrostatic interactions, perhaps due to a difference in the pKa values of bound ATP and ADP, could play a major role in the structural change induced by ATP hydrolysis. Interdomain electrostatic interactions also appear to play a role in stimulation of ATPase activity due to J-domain binding and substrate binding by Hsc70. PMID:15273304

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

PubMed Central

Hong, Zebin; De Meulemeester, Laura; Jacobi, Annemarie; Pedersen, Jan Skov; Morth, J. Preben; Andreasen, Peter A.; Jensen, Jan K.

2016-01-01

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain). PMID:27189939

Oligosaccharide Binding in Escherichia coli Glycogen Synthase

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

Sheng, Fang; Yep, Alejandra; Feng, Lei

2010-11-17

Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of themore » enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.« less

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1: FUNCTIONAL INTERACTIONS BETWEEN THE KUNITZ-TYPE INHIBITOR DOMAIN-1 AND THE NEIGHBORING POLYCYSTIC KIDNEY DISEASE-LIKE DOMAIN.

PubMed

Hong, Zebin; De Meulemeester, Laura; Jacobi, Annemarie; Pedersen, Jan Skov; Morth, J Preben; Andreasen, Peter A; Jensen, Jan K

2016-07-01

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Insight into structural rearrangements and interdomain interactions related to electron transfer between flavin mononucleotide and heme in nitric oxide synthase: A molecular dynamics study.

PubMed

Sheng, Yinghong; Zhong, Linghao; Guo, Dahai; Lau, Gavin; Feng, Changjian

2015-12-01

Calmodulin (CaM) binding to nitric oxide synthase (NOS) enables a conformational change, in which the FMN domain shuttles between the FAD and heme domains to deliver electrons to the active site heme center. A clear understanding of this large conformational change is critical, since this step is the rate-limiting in NOS catalysis. Herein molecular dynamics simulations were conducted on a model of an oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS). This is to investigate the structural rearrangements and the domain interactions related to the FMN-heme interdomain electron transfer (IET). We carried out simulations on the iNOS oxyFMN·CaM complex models in [Fe(III)][FMNH(-)] and [Fe(II)][FMNH] oxidation states, the pre- and post-IET states. The comparison of the dynamics and conformations of the iNOS construct at the two oxidation states has allowed us to identify key factors related to facilitating the FMN-heme IET process. The computational results demonstrated, for the first time, that the conformational change is redox-dependent. Predictions of the key interacting sites in optimal interdomain FMN/heme docking are well supported by experimental data in the literature. An intra-subunit pivot region is predicted to modulate the FMN domain motion and correlate with existence of a bottleneck in the conformational sampling that leads to the electron transfer-competent state. Interactions of the residues identified in this work are proposed to ensure that the FMN domain moves with appropriate degrees of freedom and docks to proper positions at the heme domain, resulting in efficient IET and nitric oxide production. Copyright © 2015 Elsevier Inc. All rights reserved.

Policy-Based Middleware for QoS Management and Signaling in the Evolved Packet System

NASA Astrophysics Data System (ADS)

Good, Richard; Gouveia, Fabricio; Magedanz, Thomas; Ventura, Neco

The 3GPP are currently finalizing their Evolved Packet System (EPS) with the Evolved Packet Core (EPC) central to this framework. The EPC is a simplified, flat, all IP-based architecture that supports mobility between heterogeneous access networks and incorporates an evolved QoS concept based on the 3GPP Policy Control and Charging (PCC) framework. The IP Multimedia Subsystem (IMS) is an IP service element within the EPS, introduced for the rapid provisioning of innovative multimedia services. The evolved PCC framework extends the scope of operation and defines new interactions - in particular the S9 reference point is introduced to facilitate inter-domain PCC communication. This paper proposes an enhancement to the IMS/PCC framework that uses SIP routing information to discover signaling and media paths. This mechanism uses standardized IMS/PCC operations and allows applications to effectively issue resource requests from their home domain enabling QoS-connectivity across multiple domains. Because the mechanism operates at the service control layer it does not require any significant transport layer modifications or the sharing of potentially sensitive internal topology information. The evolved PCC architecture and inter-domain route discovery mechanisms were implemented in an evaluation testbed and performed favorably without adversely effecting end user experience.

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity.

PubMed

Le Coq, Johanne; Camacho-Artacho, Marta; Velázquez, José Vicente; Santiveri, Clara M; Gallego, Luis Heredia; Campos-Olivas, Ramón; Dölker, Nicole; Lietha, Daniel

2017-08-09

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P 3 . Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions.

Weakly and strongly coupled Belousov-Zhabotinsky patterns.

PubMed

Weiss, Stephan; Deegan, Robert D

2017-02-01

We investigate experimentally and numerically the synchronization of two-dimensional spiral wave patterns in the Belousov-Zhabotinsky reaction due to point-to-point coupling of two separate domains. Different synchronization modalities appear depending on the coupling strength and the initial patterns in each domain. The behavior as a function of the coupling strength falls into two qualitatively different regimes. The weakly coupled regime is characterized by inter-domain interactions that distorted but do not break wave fronts. Under weak coupling, spiral cores are pushed around by wave fronts in the other domain, resulting in an effective interaction between cores in opposite domains. In the case where each domain initially contains a single spiral, the cores form a bound pair and orbit each other at quantized distances. When the starting patterns consist of multiple randomly positioned spiral cores, the number of cores decreases with time until all that remains are a few cores that are synchronized with a partner in the other domain. The strongly coupled regime is characterized by interdomain interactions that break wave fronts. As a result, the wave patterns in both domains become identical.

Weakly and strongly coupled Belousov-Zhabotinsky patterns

NASA Astrophysics Data System (ADS)

Weiss, Stephan; Deegan, Robert D.

2017-02-01

We investigate experimentally and numerically the synchronization of two-dimensional spiral wave patterns in the Belousov-Zhabotinsky reaction due to point-to-point coupling of two separate domains. Different synchronization modalities appear depending on the coupling strength and the initial patterns in each domain. The behavior as a function of the coupling strength falls into two qualitatively different regimes. The weakly coupled regime is characterized by inter-domain interactions that distorted but do not break wave fronts. Under weak coupling, spiral cores are pushed around by wave fronts in the other domain, resulting in an effective interaction between cores in opposite domains. In the case where each domain initially contains a single spiral, the cores form a bound pair and orbit each other at quantized distances. When the starting patterns consist of multiple randomly positioned spiral cores, the number of cores decreases with time until all that remains are a few cores that are synchronized with a partner in the other domain. The strongly coupled regime is characterized by interdomain interactions that break wave fronts. As a result, the wave patterns in both domains become identical.

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity

PubMed Central

Le Coq, Johanne; Camacho-Artacho, Marta; Velázquez, José Vicente; Santiveri, Clara M; Gallego, Luis Heredia; Campos-Olivas, Ramón; Dölker, Nicole; Lietha, Daniel

2017-01-01

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions. DOI: http://dx.doi.org/10.7554/eLife.26640.001 PMID:28792888

The flexibility and dynamics of protein disulfide isomerase

PubMed Central

Wells, Stephen A.; Emilio Jimenez‐Roldan, J.; Bhattacharyya, Moitrayee; Vishweshwara, Saraswathi; Freedman, Robert B.

2016-01-01

ABSTRACT We have studied the mobility of the multidomain folding catalyst, protein disulfide isomerase (PDI), by a coarse‐graining approach based on flexibility. We analyze our simulations of yeast PDI (yPDI) using measures of backbone movement, relative positions and orientations of domains, and distances between functional sites. We find that there is interdomain flexibility at every interdomain junction but these show very different characteristics. The extent of interdomain flexibility is such that yPDI's two active sites can approach much more closely than is found in crystal structures—and indeed hinge motion to bring these sites into proximity is the lowest energy normal mode of motion of the protein. The flexibility predicted for yPDI (based on one structure) includes the other known conformation of yPDI and is consistent with (i) the mobility observed experimentally for mammalian PDI and (ii) molecular dynamics. We also observe intradomain flexibility and clear differences between the domains in their propensity for internal motion. Our results suggest that PDI flexibility enables it to interact with many different partner molecules of widely different sizes and shapes, and highlights considerable similarities of yPDI and mammalian PDI. Proteins 2016; 84:1776–1785. © 2016 Wiley Periodicals, Inc. PMID:27616289

Compact Conformations of Human Protein Disulfide Isomerase

PubMed Central

Cui, Lei; Ding, Xiang; Niu, Lili; Yang, Fuquan; Wang, Chao; Wang, Chih-chen; Lou, Jizhong

2014-01-01

Protein disulfide isomerase (PDI) composed of four thioredoxin-like domains a, b, b', and a', is a key enzyme catalyzing oxidative protein folding in the endoplasmic reticulum. Large scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI) in the oxidized and reduced states were performed. The results indicate that hPDI adopts more compact conformations in solution than in the crystal structures, which are stabilized primarily by inter-domain interactions, including the salt bridges between domains a and b' observed for the first time. A prominent feature of the compact conformations is that the two catalytic domains a and a' can locate close enough for intra-molecular electron transfer, which was confirmed by the characterization of an intermediate with a disulfide between the two domains. Mutations, which disrupt the inter-domain interactions, lead to decreased reductase activity of hPDI. Our molecular dynamics simulations and biochemical experiments reveal the intrinsic conformational dynamics of hPDI and its biological impact. PMID:25084354

Segment swapping aided the evolution of enzyme function: The case of uroporphyrinogen III synthase.

PubMed

Szilágyi, András; Györffy, Dániel; Závodszky, Péter

2017-01-01

In an earlier study, we showed that two-domain segment-swapped proteins can evolve by domain swapping and fusion, resulting in a protein with two linkers connecting its domains. We proposed that a potential evolutionary advantage of this topology may be the restriction of interdomain motions, which may facilitate domain closure by a hinge-like movement, crucial for the function of many enzymes. Here, we test this hypothesis computationally on uroporphyrinogen III synthase, a two-domain segment-swapped enzyme essential in porphyrin metabolism. To compare the interdomain flexibility between the wild-type, segment-swapped enzyme (having two interdomain linkers) and circular permutants of the same enzyme having only one interdomain linker, we performed geometric and molecular dynamics simulations for these species in their ligand-free and ligand-bound forms. We find that in the ligand-free form, interdomain motions in the wild-type enzyme are significantly more restricted than they would be with only one interdomain linker, while the flexibility difference is negligible in the ligand-bound form. We also estimated the entropy costs of ligand binding associated with the interdomain motions, and find that the change in domain connectivity due to segment swapping results in a reduction of this entropy cost, corresponding to ∼20% of the total ligand binding free energy. In addition, the restriction of interdomain motions may also help the functional domain-closure motion required for catalysis. This suggests that the evolution of the segment-swapped topology facilitated the evolution of enzyme function for this protein by influencing its dynamic properties. Proteins 2016; 85:46-53. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

DOMMINO 2.0: integrating structurally resolved protein-, RNA-, and DNA-mediated macromolecular interactions

PubMed Central

Kuang, Xingyan; Dhroso, Andi; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

2016-01-01

Macromolecular interactions are formed between proteins, DNA and RNA molecules. Being a principle building block in macromolecular assemblies and pathways, the interactions underlie most of cellular functions. Malfunctioning of macromolecular interactions is also linked to a number of diseases. Structural knowledge of the macromolecular interaction allows one to understand the interaction’s mechanism, determine its functional implications and characterize the effects of genetic variations, such as single nucleotide polymorphisms, on the interaction. Unfortunately, until now the interactions mediated by different types of macromolecules, e.g. protein–protein interactions or protein–DNA interactions, are collected into individual and unrelated structural databases. This presents a significant obstacle in the analysis of macromolecular interactions. For instance, the homogeneous structural interaction databases prevent scientists from studying structural interactions of different types but occurring in the same macromolecular complex. Here, we introduce DOMMINO 2.0, a structural Database Of Macro-Molecular INteractiOns. Compared to DOMMINO 1.0, a comprehensive database on protein-protein interactions, DOMMINO 2.0 includes the interactions between all three basic types of macromolecules extracted from PDB files. DOMMINO 2.0 is automatically updated on a weekly basis. It currently includes ∼1 040 000 interactions between two polypeptide subunits (e.g. domains, peptides, termini and interdomain linkers), ∼43 000 RNA-mediated interactions, and ∼12 000 DNA-mediated interactions. All protein structures in the database are annotated using SCOP and SUPERFAMILY family annotation. As a result, protein-mediated interactions involving protein domains, interdomain linkers, C- and N- termini, and peptides are identified. Our database provides an intuitive web interface, allowing one to investigate interactions at three different resolution levels: whole subunit network, binary interaction and interaction interface. Database URL: http://dommino.org PMID:26827237

Domain size polydispersity effects on the structural and dynamical properties in lipid monolayers with phase coexistence

NASA Astrophysics Data System (ADS)

Rufeil-Fiori, Elena; Banchio, Adolfo J.

Lipid monolayers with phase coexistence are a frequently used model for lipid membranes. In these systems, domains of the liquid-condensed phase always present size polydispersity. However, very few theoretical works consider size distribution effects on the monolayer properties. Because of the difference in surface densities, domains have excess dipolar density with respect to the surrounding liquid expanded phase, originating a dipolar inter-domain interaction. This interaction depends on the domain area, and hence the presence of a domain size distribution is associated with interaction polydispersity. Inter-domain interactions are fundamental to understanding the structure and dynamics of the monolayer. For this reason, it is expected that polydispersity significantly alters monolayer properties. By means of Brownian dynamics simulations, we study the radial distribution function (RDF), the average mean square displacement and the average time-dependent self-diffusion coefficient, D(t), of lipid monolayers with normal distributed size domains. It was found that polydispersity strongly affects the value of the interaction strength obtained, which is greatly underestimated if polydispersity is not considered. However, within a certain range of parameters, the RDF obtained from a polydisperse model can be well approximated by that of a monodisperse model, suitably fitting the interaction strength, even for 40% polydispersities. For small interaction strengths or small polydispersities, the polydisperse systems obtained from fitting the experimental RDF have an average mean square displacement and D(t) in good agreement with that of the monodisperse system.

Inter-Domain Roaming Mechanism Transparent to Mobile Nodes among PMIPv6 Networks

NASA Astrophysics Data System (ADS)

Park, Soochang; Lee, Euisin; Jin, Min-Sook; Kim, Sang-Ha

In Proxy Mobile IPv6 (PMIPv6), when a Mobile Node (MN) enters a PMIPv6 domain and attaches to an access link, the router on the access link detects attachment of the MN by the link-layer access. All elements of PMIPv6 including the router then provide network-based mobility management service for the MN. If the MN moves to another router in this PMIPv6 domain, the new router emulates attachment to the previous router by providing same network prefix to the MN. In other words, PMIPv6 provides rapid mobility management based on layer-2 attachment and transparent mobility support to the MN by emulating layer-3 attachment with respect to intra-domain roaming. However, when the MN moves to other PMIPv6 domains, although the domains also provide the network-based mobility management service, the MN should exploit the host-based mobility management protocol, i.e. Mobile IPv6 (MIPv6), for the inter-domain roaming. Hence, this letter proposes the rapid and transparent inter-domain roaming mechanism controlled by the networks adopting PMIPv6.

Elucidating nitric oxide synthase domain interactions by molecular dynamics.

PubMed

Hollingsworth, Scott A; Holden, Jeffrey K; Li, Huiying; Poulos, Thomas L

2016-02-01

Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing L-Arg to NO and L-citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH-derived electrons are transferred to the heme-containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN-to-heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme-FMN-calmodulin NOS complex based on the recent biophysical studies using a 105-ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis. © 2015 The Protein Society.

Domain size polydispersity effects on the structural and dynamical properties in lipid monolayers with phase coexistence.

PubMed

Rufeil-Fiori, Elena; Banchio, Adolfo J

2018-03-07

In lipid monolayers with phase coexistence, domains of the liquid-condensed phase always present size polydispersity. However, very few theoretical works consider size distribution effects on the monolayer properties. Because of the difference in surface densities, domains have excess dipolar density with respect to the surrounding liquid expanded phase, originating a dipolar inter-domain interaction. This interaction depends on the domain area, and hence the presence of a domain size distribution is associated with interaction polydispersity. Inter-domain interactions are fundamental to understanding the structure and dynamics of the monolayer. For this reason, it is expected that polydispersity significantly alters monolayer properties. By means of Brownian dynamics simulations, we study the radial distribution function (RDF), the average mean square displacement and the average time-dependent self-diffusion coefficient, D(t), of lipid monolayers with normally distributed size domains. For this purpose, we vary the relevant system parameters, polydispersity and interaction strength, within a range of experimental interest. We also analyze the consequences of using a monodisperse model to determine the interaction strength from an experimental RDF. We find that polydispersity strongly affects the value of the interaction strength, which is greatly underestimated if polydispersity is not considered. However, within a certain range of parameters, the RDF obtained from a polydisperse model can be well approximated by that of a monodisperse model, by suitably fitting the interaction strength, even for 40% polydispersities. For small interaction strengths or small polydispersities, the polydisperse systems obtained from fitting the experimental RDF have an average mean square displacement and D(t) in good agreement with that of the monodisperse system.

Recent events dominate interdomain lateral gene transfers between prokaryotes and eukaryotes and, with the exception of endosymbiotic gene transfers, few ancient transfer events persist

PubMed Central

Katz, Laura A.

2015-01-01

While there is compelling evidence for the impact of endosymbiotic gene transfer (EGT; transfer from either mitochondrion or chloroplast to the nucleus) on genome evolution in eukaryotes, the role of interdomain transfer from bacteria and/or archaea (i.e. prokaryotes) is less clear. Lateral gene transfers (LGTs) have been argued to be potential sources of phylogenetic information, particularly for reconstructing deep nodes that are difficult to recover with traditional phylogenetic methods. We sought to identify interdomain LGTs by using a phylogenomic pipeline that generated 13 465 single gene trees and included up to 487 eukaryotes, 303 bacteria and 118 archaea. Our goals include searching for LGTs that unite major eukaryotic clades, and describing the relative contributions of LGT and EGT across the eukaryotic tree of life. Given the difficulties in interpreting single gene trees that aim to capture the approximately 1.8 billion years of eukaryotic evolution, we focus on presence–absence data to identify interdomain transfer events. Specifically, we identify 1138 genes found only in prokaryotes and representatives of three or fewer major clades of eukaryotes (e.g. Amoebozoa, Archaeplastida, Excavata, Opisthokonta, SAR and orphan lineages). The majority of these genes have phylogenetic patterns that are consistent with recent interdomain LGTs and, with the notable exception of EGTs involving photosynthetic eukaryotes, we detect few ancient interdomain LGTs. These analyses suggest that LGTs have probably occurred throughout the history of eukaryotes, but that ancient events are not maintained unless they are associated with endosymbiotic gene transfer among photosynthetic lineages. PMID:26323756

An interdomain network: the endobacterium of a mycorrhizal fungus promotes antioxidative responses in both fungal and plant hosts.

PubMed

Vannini, Candida; Carpentieri, Andrea; Salvioli, Alessandra; Novero, Mara; Marsoni, Milena; Testa, Lorenzo; de Pinto, Maria Concetta; Amoresano, Angela; Ortolani, Francesca; Bracale, Marcella; Bonfante, Paola

2016-07-01

Arbuscular mycorrhizal fungi (AMF) are obligate plant biotrophs that may contain endobacteria in their cytoplasm. Genome sequencing of Candidatus Glomeribacter gigasporarum revealed a reduced genome and dependence on the fungal host. RNA-seq analysis of the AMF Gigaspora margarita in the presence and absence of the endobacterium indicated that endobacteria have an important role in the fungal pre-symbiotic phase by enhancing fungal bioenergetic capacity. To improve the understanding of fungal-endobacterial interactions, iTRAQ (isobaric tags for relative and absolute quantification) quantitative proteomics was used to identify differentially expressed proteins in G. margarita germinating spores with endobacteria (B+), without endobacteria in the cured line (B-) and after application of the synthetic strigolactone GR24. Proteomic, transcriptomic and biochemical data identified several fungal and bacterial proteins involved in interspecies interactions. Endobacteria influenced fungal growth, calcium signalling and metabolism. The greatest effects were on fungal primary metabolism and respiration, which was 50% higher in B+ than in B-. A shift towards pentose phosphate metabolism was detected in B-. Quantification of carbonylated proteins indicated that the B- line had higher oxidative stress levels, which were also observed in two host plants. This study shows that endobacteria generate a complex interdomain network that affects AMF and fungal-plant interactions. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

PubMed Central

Wang, Tao; Li, Hua; Lin, Gang; Tang, Chunyan; Li, Dongyang; Nathan, Carl; Darwin, K. Heran; Li, Huilin

2009-01-01

Summary Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPγS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved inter-domain showed a five-stranded double β-barrel structure containing a Greek key motif. The structure and mutagenesis indicate a major role of the inter-domain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome. PMID:19836337

Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

PubMed Central

Eissenberg, J C; Ayyagari, R; Gomes, X V; Burgers, P M

1997-01-01

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks. PMID:9343398

Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

PubMed

Sempombe, Joseph; Elmore, Bradley O; Sun, Xi; Dupont, Andrea; Ghosh, Dipak K; Guillemette, J Guy; Kirk, Martin L; Feng, Changjian

2009-05-27

The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1.

PubMed

Vercoulen, Yvonne; Kondo, Yasushi; Iwig, Jeffrey S; Janssen, Axel B; White, Katharine A; Amini, Mojtaba; Barber, Diane L; Kuriyan, John; Roose, Jeroen P

2017-09-27

RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

Voltage-sensing phosphatase modulation by a C2 domain.

PubMed

Castle, Paul M; Zolman, Kevin D; Kohout, Susy C

2015-01-01

The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in VSP function.

Voltage-sensing phosphatase modulation by a C2 domain

PubMed Central

Castle, Paul M.; Zolman, Kevin D.; Kohout, Susy C.

2015-01-01

The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in VSP function. PMID:25904865

The N-terminal Region of the Ubiquitin Regulatory X (UBX) Domain-containing Protein 1 (UBXD1) Modulates Interdomain Communication within the Valosin-containing Protein p97*

PubMed Central

Trusch, Franziska; Matena, Anja; Vuk, Maja; Koerver, Lisa; Knævelsrud, Helene; Freemont, Paul S.; Meyer, Hemmo; Bayer, Peter

2015-01-01

Valosin-containing protein/p97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of p97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the p97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/p97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for p97. In reverse titration experiments, we identified two distant epitopes on the p97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of p97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to p97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the p97-ND1 interface, thereby modulating interdomain communication of p97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of p97 cofactors. PMID:26475856

Modulation and Functional Role of the Orientations of the N- and P-Domains of Cu+ -Transporting ATPase along the Ion Transport Cycle.

PubMed

Meng, Dan; Bruschweiler-Li, Lei; Zhang, Fengli; Brüschweiler, Rafael

2015-08-18

Ion transport of different P-type ATPases is regulated similarly through the interplay of multiple protein domains. In the presence of ATP, binding of a cation to the ion binding site in the transmembrane helices leads to the phosphorylation of the P-domain, allowing ion transfer across the membrane. The details of the mechanism, however, are not clear. Here, we report the modulation of the orientation between the N- and P-domains of Cu(+)-transporting ATPase along the ion transport cycle using high-resolution nuclear magnetic resonance spectroscopy in solution. On the basis of residual dipolar coupling measurements, it is found that the interdomain orientation (relative openness) of the N- and P-domains is distinctly modulated depending on the specific state of the N- and P-domains along the ion translocation cycle. The two domains' relative position in the apo state is semiopen, whereas it becomes closed upon binding of ATP to the N-domain. After phosphorylation of the P-domain and the release of ADP, the opening, however, becomes the widest among all the states. We reason such wide opening resulting from the departure of ADP prepares the N- and P-domains to accommodate the A-domain for interaction and, hence, promote ion transport and allow dephosphorylation of the P-domain. Such wide interdomain opening is abolished when an Asn to Asp mutation is introduced into the conserved DXXK motif located in the hinge region of the N- and P-domains of Cu(+)-ATPase, suggesting the indispensible role of the N- and P-interdomain orientation during ion transportation. Our results shed new light on the structural and mechanistic details of P-type ATPase function at large.

Multi-Domain SDN Survivability for Agricultural Wireless Sensor Networks.

PubMed

Huang, Tao; Yan, Siyu; Yang, Fan; Liu, Jiang

2016-11-06

Wireless sensor networks (WSNs) have been widely applied in agriculture field; meanwhile, the advent of multi-domain software-defined networks (SDNs) have improved the wireless resource utilization rate and strengthened network management. In recent times, multi-domain SDNs have been applied to agricultural sensor networks, namely multi-domain software-defined wireless sensor networks (SDWSNs). However, when the SDNs controlling agriculture networks suddenly become unavailable, whether intra-domain or inter-domain, sensor network communication is abnormal because of the loss of control. Moreover, there are controller and switch info-updating problems even if the controller becomes available again. To resolve these problems, this paper proposes a new approach based on an Open vSwitch extension for multi-domain SDWSNs, which can enhance agriculture network survivability and stability. We achieved this by designing a connection-state mechanism, a communication mechanism on both L2 and L3, and an info-updating mechanism based on Open vSwitch. The experimental results show that, whether it is agricultural inter-domain or intra-domain during the controller failure period, the sensor switches can enter failure recovery mode as soon as possible so that the sensor network keeps a stable throughput, a short failure recovery time below 300 ms, and low packet loss. Further, the domain can smoothly control the domain network again once the controller becomes available. This approach based on an Open vSwitch extension can enhance the survivability and stability of multi-domain SDWSNs in precision agriculture.

Multi-Domain SDN Survivability for Agricultural Wireless Sensor Networks

PubMed Central

Huang, Tao; Yan, Siyu; Yang, Fan; Liu, Jiang

2016-01-01

Wireless sensor networks (WSNs) have been widely applied in agriculture field; meanwhile, the advent of multi-domain software-defined networks (SDNs) have improved the wireless resource utilization rate and strengthened network management. In recent times, multi-domain SDNs have been applied to agricultural sensor networks, namely multi-domain software-defined wireless sensor networks (SDWSNs). However, when the SDNs controlling agriculture networks suddenly become unavailable, whether intra-domain or inter-domain, sensor network communication is abnormal because of the loss of control. Moreover, there are controller and switch info-updating problems even if the controller becomes available again. To resolve these problems, this paper proposes a new approach based on an Open vSwitch extension for multi-domain SDWSNs, which can enhance agriculture network survivability and stability. We achieved this by designing a connection-state mechanism, a communication mechanism on both L2 and L3, and an info-updating mechanism based on Open vSwitch. The experimental results show that, whether it is agricultural inter-domain or intra-domain during the controller failure period, the sensor switches can enter failure recovery mode as soon as possible so that the sensor network keeps a stable throughput, a short failure recovery time below 300 ms, and low packet loss. Further, the domain can smoothly control the domain network again once the controller becomes available. This approach based on an Open vSwitch extension can enhance the survivability and stability of multi-domain SDWSNs in precision agriculture. PMID:27827971

Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication

PubMed Central

Stetz, Gabrielle; Verkhivker, Gennady M.

2017-01-01

Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we introduced a community-hopping model of allosteric communication. Atomistic reconstruction of signaling pathways in the DnaK structures captured a direction-specific mechanism and molecular details of signal transmission that are fully consistent with the mutagenesis experiments. The results of our study reconciled structural and functional experiments from a network-centric perspective by showing that global properties of the residue interaction networks and coevolutionary signatures may be linked with specificity and diversity of allosteric regulation mechanisms. PMID:28095400

Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2017-01-01

Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we introduced a community-hopping model of allosteric communication. Atomistic reconstruction of signaling pathways in the DnaK structures captured a direction-specific mechanism and molecular details of signal transmission that are fully consistent with the mutagenesis experiments. The results of our study reconciled structural and functional experiments from a network-centric perspective by showing that global properties of the residue interaction networks and coevolutionary signatures may be linked with specificity and diversity of allosteric regulation mechanisms.

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

PubMed Central

Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

2009-01-01

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

Structural Overview of the Nuclear Receptor Superfamily: Insights into Physiology and Therapeutics

PubMed Central

Huang, Pengxiang; Chandra, Vikas; Rastinejad, Fraydoon

2013-01-01

As ligand-regulated transcription factors, the nuclear hormone receptors are nearly ideal drug targets, with internal pockets that bind to hydrophobic, drug-like molecules and well-characterized ligand-induced conformational changes that recruit transcriptional coregulators to promoter elements. Yet, due to the multitude of genes under the control of a single receptor, the major challenge has been the identification of ligands with gene-selective actions, impacting disease outcomes through a narrow subset of target genes and not across their entire gene-regulatory repertoire. Here, we summarize the concepts and work to date underlying the development of steroidal and nonsteroidal receptor ligands, including the use of crystal structures, high-throughput screens, and rational design approaches for finding useful therapeutic molecules. Difficulties in finding selective receptor modulators require a more complete understanding of receptor interdomain communications, posttranslational modifications, and receptor-protein interactions that could be exploited for target gene selectivity. PMID:20148675

Mutation-Linked Defective Inter-Domain Interactions within Ryanodine Receptor Cause Aberrant Ca2+ Release Leading to Catecholaminergic Polymorphic Ventricular Tachycardia

PubMed Central

Suetomi, Takeshi; Yano, Masafumi; Uchinoumi, Hitoshi; Fukuda, Masakazu; Hino, Akihiro; Ono, Makoto; Xu, Xiaojuan; Tateishi, Hiroki; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Ikeda, Yasuhiho; Yamamoto, Takeshi; Ikemoto, Noriaki; Matsuzaki, Masunori

2011-01-01

Background The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia (CPVT) is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. Here, we investigated mutation-induced conformational defects of RyR2 using a knock-in (KI) mouse model expressing the human CPVT-associated RyR2 mutant (S2246L; Serine to Leucine mutation at the residue 2246). Methods and Results All KI mice we examined produced VT after exercise on a treadmill. cAMP-dependent increase in the frequency of Ca2+ sparks was more pronounced in saponin-permeabilized KI cardiomyocytes than in WT cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232–2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741– 2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding. Fluorescence quench assays of inter-domain interactions within RyR2 showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1-600)/central (2000–2500) domain pair in an allosteric manner. Dantrolene corrected the mutation-caused domain unzipping of the domain switch, and stopped the exercise-induced ventricular tachycardia. Conclusions The CPVT-linked mutation of RyR2, S2246L, causes an abnormally tight local sub-domain/sub-domain interaction within the central domain involving the mutation site, which induces defective interaction between the N-terminal and central domains. This results in an erroneous activation of Ca2+ channel in a diastolic state reflecting on the increased Ca2+ spark frequency, which then leads to lethal arrhythmia. PMID:21768539

Mutation-linked defective interdomain interactions within ryanodine receptor cause aberrant Ca²⁺release leading to catecholaminergic polymorphic ventricular tachycardia.

PubMed

Suetomi, Takeshi; Yano, Masafumi; Uchinoumi, Hitoshi; Fukuda, Masakazu; Hino, Akihiro; Ono, Makoto; Xu, Xiaojuan; Tateishi, Hiroki; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Ikeda, Yasuhiro; Yamamoto, Takeshi; Ikemoto, Noriaki; Matsuzaki, Masunori

2011-08-09

The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. In the present study, we investigated mutation-induced conformational defects of RyR2 using a knockin mouse model expressing the human catecholaminergic polymorphic ventricular tachycardia-associated RyR2 mutant (S2246L; serine to leucine mutation at the residue 2246). All knockin mice we examined produced ventricular tachycardia after exercise on a treadmill. cAMP-dependent increase in the frequency of Ca²⁺ sparks was more pronounced in saponin-permeabilized knockin cardiomyocytes than in wild-type cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232 to 2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741 to 2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding. Fluorescence quench assays of interdomain interactions within RyR2 showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1 to 600)/central (2000 to 2500) domain pair in an allosteric manner. Dantrolene corrected the mutation-caused domain unzipping of the domain switch and stopped the exercise-induced ventricular tachycardia. The catecholaminergic polymorphic ventricular tachycardia-linked mutation of RyR2, S2246L, causes an abnormally tight local subdomain-subdomain interaction within the central domain involving the mutation site, which induces defective interaction between the N-terminal and central domains. This results in an erroneous activation of Ca²⁺ channel in a diastolic state reflecting on the increased Ca²⁺ spark frequency, which then leads to lethal arrhythmia.

Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

NASA Astrophysics Data System (ADS)

Medina-Carmona, Encarnación; Neira, Jose L.; Salido, Eduardo; Fuchs, Julian E.; Palomino-Morales, Rogelio; Timson, David J.; Pey, Angel L.

2017-03-01

Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

PubMed Central

Koenig, Patrick; Lee, Chingwei V.; Walters, Benjamin T.; Janakiraman, Vasantharajan; Stinson, Jeremy; Patapoff, Thomas W.; Fuh, Germaine

2017-01-01

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. PMID:28057863

A Domain Decomposition Parallelization of the Fast Marching Method

NASA Technical Reports Server (NTRS)

Herrmann, M.

2003-01-01

In this paper, the first domain decomposition parallelization of the Fast Marching Method for level sets has been presented. Parallel speedup has been demonstrated in both the optimal and non-optimal domain decomposition case. The parallel performance of the proposed method is strongly dependent on load balancing separately the number of nodes on each side of the interface. A load imbalance of nodes on either side of the domain leads to an increase in communication and rollback operations. Furthermore, the amount of inter-domain communication can be reduced by aligning the inter-domain boundaries with the interface normal vectors. In the case of optimal load balancing and aligned inter-domain boundaries, the proposed parallel FMM algorithm is highly efficient, reaching efficiency factors of up to 0.98. Future work will focus on the extension of the proposed parallel algorithm to higher order accuracy. Also, to further enhance parallel performance, the coupling of the domain decomposition parallelization to the G(sub 0)-based parallelization will be investigated.

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

Lewis, Hal A.; Zhao, Xun; Wang, Chi

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformationmore » but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.« less

Regulation of adhesion behavior of murine macrophage using supported lipid membranes displaying tunable mannose domains

NASA Astrophysics Data System (ADS)

Kaindl, T.; Oelke, J.; Pasc, A.; Kaufmann, S.; Konovalov, O. V.; Funari, S. S.; Engel, U.; Wixforth, A.; Tanaka, M.

2010-07-01

Highly uniform, strongly correlated domains of synthetically designed lipids can be incorporated into supported lipid membranes. The systematic characterization of membranes displaying a variety of domains revealed that the equilibrium size of domains significantly depends on the length of fluorocarbon chains, which can be quantitatively interpreted within the framework of an equivalent dipole model. A mono-dispersive, narrow size distribution of the domains enables us to treat the inter-domain correlations as two-dimensional colloidal crystallization and calculate the potentials of mean force. The obtained results demonstrated that both size and inter-domain correlation can precisely be controlled by the molecular structures. By coupling α-D-mannose to lipid head groups, we studied the adhesion behavior of the murine macrophage (J774A.1) on supported membranes. Specific adhesion and spreading of macrophages showed a clear dependence on the density of functional lipids. The obtained results suggest that such synthetic lipid domains can be used as a defined platform to study how cells sense the size and distribution of functional molecules during adhesion and spreading.

Interdomain flexibility and pH-induced conformational changes of AcrA revealed by molecular dynamics simulations.

PubMed

Wang, Beibei; Weng, Jingwei; Fan, Kangnian; Wang, Wenning

2012-03-15

The membrane fusion protein (MFP) AcrA is proposed to link the inner membrane transporter AcrB and outer membrane protein TolC, forming the tripartite AcrAB-TolC efflux pump, and was shown to be functionally indispensible. Structural and EPR studies showed that AcrA has high conformational flexibility and exhibited pH-induced conformational change. In this study, we built the complete structure of AcrA through homology modeling and performed atomistic simulations of AcrA at different pH values. It was shown that the conformational flexibility of AcrA originates from the motions of α-hairpin and MP domains. The conformational dynamics of AcrA is sensitive to specific point mutations and pH values. In agreement with the EPR experiments, the interdomain motions were restrained upon lowering pH from 7.0 to 5.0 in the simulations. It was found that the protonation/deprotonation of His285 underlies the pH-regulated conformational dynamics of AcrA by disturbing the local hydrogen bond interactions, suggesting that the changes of pH in the periplasm accompanying the drug efflux could act as a signal to trigger the action of AcrA, which undergoes reversible conformational rearrangement. © 2012 American Chemical Society

Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners

PubMed Central

Felicori, Liza; Jameson, Katie H.; Roblin, Pierre; Fogg, Mark J.; Garcia-Garcia, Transito; Ventroux, Magali; Cherrier, Mickaël V.; Bazin, Alexandre; Noirot, Philippe; Wilkinson, Anthony J.; Molina, Franck; Terradot, Laurent; Noirot-Gros, Marie-Françoise

2016-01-01

YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell. PMID:26615189

Bidomain structures formed in lithium niobate and lithium tantalate single crystals by light annealing

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

Kubasov, I. V., E-mail: kubasov.ilya@gmail.com; Kislyuk, A. M.; Bykov, A. S.

The bidomain structures produced by light external heating in z-cut lithium niobate and lithium tantalate single crystals are formed and studied. Interdomain regions about 200 and 40 μm wide in, respectively, LiNbO{sub 3} and LiTaO{sub 3} bidomain crystals are visualized and studied by optical microscopy and piezoresponse force microscopy. Extended chains and lines of domains in the form of thin layers with a width less than 10 μm in volume, which penetrate the interdomain region and spread over distances of up to 1 mm, are found.

Ultrastable cellulosome-adhesion complex tightens under load.

PubMed

Schoeler, Constantin; Malinowska, Klara H; Bernardi, Rafael C; Milles, Lukas F; Jobst, Markus A; Durner, Ellis; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Schulten, Klaus; Gaub, Hermann E; Nash, Michael A

2014-12-08

Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand-receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand-receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600-750 pN, making it one of the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components may help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass.

Enabling Secure XMPP Communications in Federated IoT Clouds Through XEP 0027 and SAML/SASL SSO

PubMed Central

Celesti, Antonio; Fazio, Maria; Villari, Massimo

2017-01-01

Nowadays, in the panorama of Internet of Things (IoT), finding a right compromise between interactivity and security is not trivial at all. Currently, most of pervasive communication technologies are designed to work locally. As a consequence, the development of large-scale Internet services and applications is not so easy for IoT Cloud providers. The main issue is that both IoT architectures and services have started as simple but they are becoming more and more complex. Consequently, the web service technology is often inappropriate. Recently, many operators in both academia and industry fields are considering the possibility to adopt the eXtensible Messaging and Presence Protocol (XMPP) for the implementation of IoT Cloud communication systems. In fact, XMPP offers many advantages in term of real-time capabilities, efficient data distribution, service discovery and inter-domain communication compared to other technologies. Nevertheless, the protocol lacks of native security, data confidentiality and trustworthy federation features. In this paper, considering an XMPP-based IoT Cloud architectural model, we discuss how can be possible to enforce message signing/encryption and Single-Sign On (SSO) authentication respectively for secure inter-module and inter-domain communications in a federated environment. Experiments prove that security mechanisms introduce an acceptable overhead, considering the obvious advantages achieved in terms of data trustiness and privacy. PMID:28178214

Enabling Secure XMPP Communications in Federated IoT Clouds Through XEP 0027 and SAML/SASL SSO.

PubMed

Celesti, Antonio; Fazio, Maria; Villari, Massimo

2017-02-07

Nowadays, in the panorama of Internet of Things (IoT), finding a right compromise between interactivity and security is not trivial at all. Currently, most of pervasive communication technologies are designed to work locally. As a consequence, the development of large-scale Internet services and applications is not so easy for IoT Cloud providers. The main issue is that both IoT architectures and services have started as simple but they are becoming more and more complex. Consequently, the web service technology is often inappropriate. Recently, many operators in both academia and industry fields are considering the possibility to adopt the eXtensible Messaging and Presence Protocol (XMPP) for the implementation of IoT Cloud communication systems. In fact, XMPP offers many advantages in term of real-time capabilities, efficient data distribution, service discovery and inter-domain communication compared to other technologies. Nevertheless, the protocol lacks of native security, data confidentiality and trustworthy federation features. In this paper, considering an XMPP-based IoT Cloud architectural model, we discuss how can be possible to enforce message signing/encryption and Single-Sign On (SSO) authentication respectively for secure inter-module and inter-domain communications in a federated environment. Experiments prove that security mechanisms introduce an acceptable overhead, considering the obvious advantages achieved in terms of data trustiness and privacy.

Long-Range Correlations and Memory in the Dynamics of Internet Interdomain Routing

PubMed Central

Havlin, Shlomo; Krioukov, Dmitri

2015-01-01

Data transfer is one of the main functions of the Internet. The Internet consists of a large number of interconnected subnetworks or domains, known as Autonomous Systems (ASes). Due to privacy and other reasons the information about what route to use to reach devices within other ASes is not readily available to any given AS. The Border Gateway Protocol (BGP) is responsible for discovering and distributing this reachability information to all ASes. Since the topology of the Internet is highly dynamic, all ASes constantly exchange and update this reachability information in small chunks, known as routing control packets or BGP updates. In the view of the quick growth of the Internet there are significant concerns with the scalability of the BGP updates and the efficiency of the BGP routing in general. Motivated by these issues we conduct a systematic time series analysis of BGP update rates. We find that BGP update time series are extremely volatile, exhibit long-term correlations and memory effects, similar to seismic time series, or temperature and stock market price fluctuations. The presented statistical characterization of BGP update dynamics could serve as a basis for validation of existing and developing better models of Internet interdomain routing. PMID:26529312

Long-Range Correlations and Memory in the Dynamics of Internet Interdomain Routing.

PubMed

Kitsak, Maksim; Elmokashfi, Ahmed; Havlin, Shlomo; Krioukov, Dmitri

2015-01-01

Data transfer is one of the main functions of the Internet. The Internet consists of a large number of interconnected subnetworks or domains, known as Autonomous Systems (ASes). Due to privacy and other reasons the information about what route to use to reach devices within other ASes is not readily available to any given AS. The Border Gateway Protocol (BGP) is responsible for discovering and distributing this reachability information to all ASes. Since the topology of the Internet is highly dynamic, all ASes constantly exchange and update this reachability information in small chunks, known as routing control packets or BGP updates. In the view of the quick growth of the Internet there are significant concerns with the scalability of the BGP updates and the efficiency of the BGP routing in general. Motivated by these issues we conduct a systematic time series analysis of BGP update rates. We find that BGP update time series are extremely volatile, exhibit long-term correlations and memory effects, similar to seismic time series, or temperature and stock market price fluctuations. The presented statistical characterization of BGP update dynamics could serve as a basis for validation of existing and developing better models of Internet interdomain routing.

Novel forms of colloidal self-organization in temporally and spatially varying external fields: from low-density network-forming fluids to spincoated crystals

NASA Astrophysics Data System (ADS)

Yethiraj, Anand

2010-03-01

External fields affect self-organization in Brownian colloidal suspensions in many different ways [1]. High-frequency time varying a.c. electric fields can induce effectively quasi-static dipolar inter-particle interactions. While dipolar interactions can provide access to multiple open equilibrium crystal structures [2] whose origin is now reasonably well understood, they can also give rise to competing interactions on short and long length scales that produce unexpected low-density ordered phases [3]. Farther from equilibrium, competing external fields are active in colloid spincoating. Drying colloidal suspensions on a spinning substrate produces a ``perfect polycrystal'' - tiny polycrystalline domains that exhibit long-range inter-domain orientational order [4] with resultant spectacular optical effects that are decoupled from single-crystallinity. High-speed movies of drying crystals yield insights into mechanisms of structure formation. Phenomena arising from multiple spatially- and temporally-varying external fields can give rise to further control of order and disorder, with potential application as patterned (photonic and magnetic) materials. [4pt] [1] A. Yethiraj, Soft Matter 3, 1099 (2007). [2] A. Yethiraj, A. van Blaaderen, Nature 421, 513 (2003). [3] A.K. Agarwal, A. Yethiraj, Phys. Rev. Lett ,102, 198301 (2009). [4] C. Arcos, K. Kumar, W. Gonz'alez-Viñas, R. Sirera, K. Poduska, A. Yethiraj, Phys. Rev. E ,77, 050402(R) (2008).

Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation

DOE PAGES

Rivera-Cancel, Giomar; Ko, Wen-huang; Tomchick, Diana R.; ...

2014-12-02

Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. In this paper, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light–oxygen–voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain.more » The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. Finally, we suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.« less

Molecular dynamics-based model of VEGF-A and its heparin interactions.

PubMed

Uciechowska-Kaczmarzyk, Urszula; Babik, Sándor; Zsila, Ferenc; Bojarski, Krzysztof Kamil; Beke-Somfai, Tamás; Samsonov, Sergey A

2018-06-01

We present a computational model of the Vascular Endothelial Growth Factor (VEGF), an important regulator of blood vessels formation, which function is affected by its heparin interactions. Although structures of a receptor binding (RBD) and a heparin binding domain (HBD) of VEGF are known, there are structural data neither on the 12 amino acids interdomain linker nor on its complexes with heparin. We apply molecular docking and molecular dynamics techniques combined with circular dichroism spectroscopy to model the full structure of the dimeric VEGF and to propose putative molecular mechanisms underlying the function of VEGF/VEGF receptors/heparin system. We show that both the conformational flexibility of the linker and the formation of HBD-heparin-HBD sandwich-like structures regulate the mutual disposition of HBDs and so affect the VEGF-mediated signalling. Copyright © 2018 Elsevier Inc. All rights reserved.

Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

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

Zhang, Z.; Swaminathan, S.; Zhou, R.

2011-02-18

Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. Themore » structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.« less

Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

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

Z Zhang; R Zhou; J Sauder

2011-12-31

Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. Themore » structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.« less

Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies.

PubMed

Xiong, Yijia; Ford, Nicole R; Hecht, Karen A; Roesijadi, Guritno; Squier, Thomas C

2017-11-15

Here, we identify the importance of molecular crowding agents in the functional stabilization of scFv antibodies. Antibodies were tethered through an engineered calmodulin (CaM)-binding peptide into a stimulus-responsive hydrogel composed of poly(ethylene glycol) (PEG)-functionalized CaM. Macromolecular crowding is modulated by transient heating, which decreases effective pore sizes. Using a fluorescent ligand bound to the scFv, frequency-domain fluorescence spectroscopy was used to assess the structural coupling between the V H and the V L domains and relationships with functional stabilization. There is minimal structural coupling between the V H and the V L domains in solution, as is apparent from the substantial rotational mobility for the bound ligand, that is suggestive of an independent mobility for the V H and the V L domains. In comparison, the hydrogel matrix acts to structurally couple the V H and the V L domains, resulting in a reduction in rotational mobility and a retention of ligand binding in the presence of 8.0 M urea. Under these same conditions, ligand binding is disrupted for scFv antibodies in solution. Increases in the stabilization of scFv antibodies in hydrogels is not simply the result of molecular crowding because decreases in pore size act to destabilize ligand binding. Rather, our results suggest that the functional stabilization of the scFv antibody within the PEG hydrogel matrix includes important factors involving protein solvation that stabilize interdomain interactions between the V H and the V L domains necessary for ligand binding.

Structure of the DBL3X-DBL4ε region of the VAR2CSA placental malaria vaccine candidate: insight into DBL domain interactions

PubMed Central

Gangnard, Stéphane; Lewit-Bentley, Anita; Dechavanne, Sébastien; Srivastava, Anand; Amirat, Faroudja; Bentley, Graham A.; Gamain, Benoît

2015-01-01

The human malaria parasite, Plasmodium falciparum, is able to evade spleen-mediated clearing from blood stream by sequestering in peripheral organs. This is due to the adhesive properties conferred by the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family exported by the parasite to the surface of infected erythrocytes. Expression of the VAR2CSA variant of PfEMP1 leads to pregnancy-associated malaria, which occurs when infected erythrocytes massively sequester in the placenta by binding to low-sulfated Chondroitin Sulfate A (CSA) present in the intervillous spaces. VAR2CSA is a 350 kDa protein that carries six Duffy-Binding Like (DBL) domains, one Cysteine-rich Inter-Domain Regions (CIDR) and several inter-domain regions. In the present paper, we report for the first time the crystal structure at 2.9 Å of a VAR2CSA double domain, DBL3X-DBL4ε, from the FCR3 strain. DBL3X and DBL4ε share a large contact interface formed by residues that are invariant or highly conserved in VAR2CSA variants, which suggests that these two central DBL domains (DBL3X-DBL4ε) contribute significantly to the structuring of the functional VAR2CSA extracellular region. We have also examined the antigenicity of peptides corresponding to exposed loop regions of the DBL4ε structure. PMID:26450557

Structure of the first representative of Pfam family PF09410 (DUF2006) reveals a structural signature of the calycin superfamily that suggests a role in lipid metabolism

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

Chiu, Hsiu-Ju; Bakolitsa, Constantina; Skerra, Arne

The first structural representative of the domain of unknown function DUF2006 family, also known as Pfam family PF09410, comprises a lipocalin-like fold with domain duplication. The finding of the calycin signature in the N-terminal domain, combined with remote sequence similarity to two other protein families (PF07143 and PF08622) implicated in isoprenoid metabolism and the oxidative stress response, support an involvement in lipid metabolism. Clusters of conserved residues that interact with ligand mimetics suggest that the binding and regulation sites map to the N-terminal domain and to the interdomain interface, respectively.

A Unified Approach to Intra-Domain Security

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

Shue, Craig A; Kalafut, Andrew J.; Gupta, Prof. Minaxi

2009-01-01

While a variety of mechanisms have been developed for securing individual intra-domain protocols, none address the issue in a holistic manner. We develop a unified framework to secure prominent networking protocols within a single domain. We begin with a secure version of the DHCP protocol, which has the additional feature of providing each host with a certificate. We then leverage these certificates to secure ARP, prevent spoofing within the domain, and secure SSH and VPN connections between the domain and hosts which have previously interacted with it locally. In doing so, we also develop an incrementally deployable public key infrastructuremore » which can later be leveraged to support inter-domain authentication.« less

Ultrastable cellulosome-adhesion complex tightens under load

PubMed Central

Schoeler, Constantin; Malinowska, Klara H.; Bernardi, Rafael C.; Milles, Lukas F.; Jobst, Markus A.; Durner, Ellis; Ott, Wolfgang; Fried, Daniel B.; Bayer, Edward A.; Schulten, Klaus; Gaub, Hermann E.; Nash, Michael A.

2014-01-01

Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand–receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand–receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600–750 pN, making it one of the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components may help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass. PMID:25482395

Novel Insights into Insect-Microbe Interactions—Role of Epigenomics and Small RNAs

PubMed Central

Kim, Dohyup; Thairu, Margaret W.; Hansen, Allison K.

2016-01-01

It has become increasingly clear that microbes form close associations with the vast majority of animal species, especially insects. In fact, an array of diverse microbes is known to form shared metabolic pathways with their insect hosts. A growing area of research in insect-microbe interactions, notably for hemipteran insects and their mutualistic symbionts, is to elucidate the regulation of this inter-domain metabolism. This review examines two new emerging mechanisms of gene regulation and their importance in host-microbe interactions. Specifically, we highlight how the incipient areas of research on regulatory “dark matter” such as epigenomics and small RNAs, can play a pivotal role in the evolution of both insect and microbe gene regulation. We then propose specific models of how these dynamic forms of gene regulation can influence insect-symbiont-plant interactions. Future studies in this area of research will give us a systematic understanding of how these symbiotic microbes and animals reciprocally respond to and regulate their shared metabolic processes. PMID:27540386

Structure of Serum Amyloid A Suggests a Mechanism for Selective Lipoprotein Binding and Functions: SAA as a Hub in Macromolecular Interaction Networks

PubMed Central

Frame, Nicholas M.; Gursky, Olga

2016-01-01

Serum amyloid A is a major acute-phase plasma protein that modulates innate immunity and cholesterol homeostasis. We combine sequence analysis with x-ray crystal structures to postulate that SAA acts as an intrinsically disordered hub mediating interactions among proteins, lipids and proteoglycans. A structural model of lipoprotein-bound SAA monomer is proposed wherein two α-helices from the N-domain form a concave hydrophobic surface that binds lipoproteins. A C-domain, connected to the N-domain via a flexible linker, binds polar/charged ligands including cell receptors, bridging them with lipoproteins and re-routing cholesterol transport. Our model is supported by the SAA cleavage in the inter-domain linker to generate the 1–76 fragment deposited in reactive amyloidosis. This model sheds new light on functions of this enigmatic protein. PMID:26918388

Preventing the Androgen Receptor N/C Interaction Delays Disease Onset in a Mouse Model of SBMA.

PubMed

Zboray, Lori; Pluciennik, Anna; Curtis, Dana; Liu, Yuhong; Berman-Booty, Lisa D; Orr, Christopher; Kesler, Cristina T; Berger, Tamar; Gioeli, Daniel; Paschal, Bryce M; Merry, Diane E

2015-12-15

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by a polyglutamine expansion in the androgen receptor (AR) and is associated with misfolding and aggregation of the mutant AR. We investigated the role of an interdomain interaction between the amino (N)-terminal FxxLF motif and carboxyl (C)-terminal AF-2 domain in a mouse model of SBMA. Male transgenic mice expressing polyQ-expanded AR with a mutation in the FxxLF motif (F23A) to prevent the N/C interaction displayed substantially improved motor function compared with N/C-intact AR-expressing mice and showed reduced pathological features of SBMA. Serine 16 phosphorylation was substantially enhanced by the F23A mutation; moreover, the protective effect of AR F23A was dependent on this phosphorylation. These results reveal an important role for the N/C interaction on disease onset in mice and suggest that targeting AR conformation could be a therapeutic strategy for patients with SBMA. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction

PubMed Central

Xu, Xiaojuan; Yano, Masafumi; Uchinoumi, Hitoshi; Hino, Akihiro; Suetomi, Takeshi; Ono, Makoto; Tateishi, Hiroki; Oda, Tetsuro; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Yamamoto, Takeshi; Ikeda, Yasuhiro; Ikemoto, Noriaki; Matsuzaki, Masunori

2010-01-01

Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S). Without added effectors, the affinity of CaM binding to the RyR2 was indistinguishable between KI and WT hearts. In response to cAMP (1 μmol/L), the RyR2 phosphorylation at Ser2808 increased in both WT and KI hearts to the same extent. However, cAMP caused a significant decrease of the CaM binding affinity in KI hearts, but the affinity was unchanged in WT. Dantrolene restored a normal level of CaM-binding affinity in the cAMP-treated KI hearts, suggesting that defective inter-domain interaction between the N-terminal domain and the central domain of the RyR2 (the target of therapeutic effect of dantrolene) is involved in the cAMP-induced reduction of the CaM binding affinity. In saponin-permeabilized cardiomyocytes, the addition of cAMP increased the frequency of spontaneous Ca2+ sparks to a significantly larger extent in KI cardiomyocytes than in WT cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca2+ sparks. In conclusion, CPVT mutation causes defective inter-domain interaction, significant reduction in the ability of CaM binding to the RyR2, spontaneous Ca2+ leak, and then lethal arrhythmia. PMID:20226167

Conserved interdomain linker promotes phase separation of the multivalent adaptor protein Nck

PubMed Central

Banjade, Sudeep; Wu, Qiong; Mittal, Anuradha; Peeples, William B.; Pappu, Rohit V.; Rosen, Michael K.

2015-01-01

The organization of membranes, the cytosol, and the nucleus of eukaryotic cells can be controlled through phase separation of lipids, proteins, and nucleic acids. Collective interactions of multivalent molecules mediated by modular binding domains can induce gelation and phase separation in several cytosolic and membrane-associated systems. The adaptor protein Nck has three SRC-homology 3 (SH3) domains that bind multiple proline-rich segments in the actin regulatory protein neuronal Wiskott-Aldrich syndrome protein (N-WASP) and an SH2 domain that binds to multiple phosphotyrosine sites in the adhesion protein nephrin, leading to phase separation. Here, we show that the 50-residue linker between the first two SH3 domains of Nck enhances phase separation of Nck/N-WASP/nephrin assemblies. Two linear motifs within this element, as well as its overall positively charged character, are important for this effect. The linker increases the driving force for self-assembly of Nck, likely through weak interactions with the second SH3 domain, and this effect appears to promote phase separation. The linker sequence is highly conserved, suggesting that the sequence determinants of the driving forces for phase separation may be generally important to Nck functions. Our studies demonstrate that linker regions between modular domains can contribute to the driving forces for self-assembly and phase separation of multivalent proteins. PMID:26553976

Differential Modulation of Functional Dynamics and Allosteric Interactions in the Hsp90-Cochaperone Complexes with p23 and Aha1: A Computational Study

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2013-01-01

Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide “molecular brakes” that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity and direct the chaperone cycle by exerting the precise control over structural stability, global movements and allosteric communications in Hsp90. PMID:23977182

The structural basis for the negative regulation of thioredoxin by thioredoxin-interacting protein

PubMed Central

Hwang, Jungwon; Suh, Hyun-Woo; Jeon, Young Ho; Hwang, Eunha; Nguyen, Loi T.; Yeom, Jeonghun; Lee, Seung-Goo; Lee, Cheolju; Kim, Kyung Jin; Kang, Beom Sik; Jeong, Jin-Ok; Oh, Tae-Kwang; Choi, Inpyo; Lee, Jie-Oh; Kim, Myung Hee

2014-01-01

The redox-dependent inhibition of thioredoxin (TRX) by thioredoxin-interacting protein (TXNIP) plays a pivotal role in various cancers and metabolic syndromes. However, the molecular mechanism of this regulation is largely unknown. Here, we present the crystal structure of the TRX–TXNIP complex and demonstrate that the inhibition of TRX by TXNIP is mediated by an intermolecular disulphide interaction resulting from a novel disulphide bond-switching mechanism. Upon binding to TRX, TXNIP undergoes a structural rearrangement that involves switching of a head-to-tail interprotomer Cys63-Cys247 disulphide between TXNIP molecules to an interdomain Cys63-Cys190 disulphide, and the formation of a de novo intermolecular TXNIP Cys247-TRX Cys32 disulphide. This disulphide-switching event unexpectedly results in a domain arrangement of TXNIP that is entirely different from those of other arrestin family proteins. We further show that the intermolecular disulphide bond between TRX and TXNIP dissociates in the presence of high concentrations of reactive oxygen species. This study provides insight into TRX and TXNIP-dependent cellular regulation. PMID:24389582

Recognition of the Xenopus ribosomal core promoter by the transcription factor xUBF involves multiple HMG box domains and leads to an xUBF interdomain interaction.

PubMed

Leblanc, B; Read, C; Moss, T

1993-02-01

The interaction of the ribosomal transcription factor xUBF with the RNA polymerase I core promoter of Xenopus laevis has been studied both at the DNA and protein levels. It is shown that a single xUBF-DNA complex forms over the 40S initiation site (+1) and involves at least the DNA sequences between -20 and +60 bp. DNA sequences upstream of +10 and downstream of +18 are each sufficient to direct complex formation independently. HMG box 1 of xUBF independently recognizes the sequences -20 to -1 and +1 to +22 and the addition of the N-terminal dimerization domain to HMG box 1 stabilizes its interaction with these sequences approximately 10-fold. HMG boxes 2/3 interact with the DNA downstream of +22 and can independently position xUBF across the initiation site. The C-terminal segment of xUBF, HMG boxes 4, 5 or the acidic domain, directly or indirectly interact with HMG box 1, making the core promoter sequences between -11 and -15 hypersensitive to DNase. This interaction also requires the DNA sequences between +17 and +32, i.e. the HMG box 2/3 binding site. The data suggest extensive folding of the core promoter within the xUBF complex.

The extended TRIP supporting VoIP routing reservation with distributed QoS

NASA Astrophysics Data System (ADS)

Wang, Furong; Wu, Ye

2004-04-01

In this paper, an existing protocol, i.e. TRIP (Telephony Routing over IP) is developed to provide distributed QoS when making resource reservations for VoIP services such as H.323, SIP. Enhanced LSs (location servers) are deployed in ITADs (IP Telephony Administrative Domains) to take in charge of intra-domain routing policy because of small propagation price. It is an easy way to find an IP telephone route for intra-domain VoIP media association and simultaneously possess intra-domain load balancing features. For those routing reservations bridging domains, inter-domain routing policy is responsible for finding the shortest inter-domain route with enough resources. I propose the routing preference policy based on QoS price when the session traffic is shaped by a token bucket, related QoS messages, and message cooperation.

Molecular dynamics simulations of human E3 ubiquitin ligase Parkin

PubMed Central

Qiu, Shi; Zhu, Shun; Xu, Shan; Han, Yanyan; Liu, Wen; Zuo, Ji

2017-01-01

Human E3 ubiquitin protein ligase parkin (Parkin) mediates mitophagy to maintain mitochondrial homeostasis. Parkin mutations are common genetic causes of early onset familial Parkinson's disease. The molecular mechanism of Parkin activation has been widely studied with emerging evidence suggesting an essential role of the phosphorylated (phospho)-ubiquitin interaction. However, the underlying mechanism of the phospho-ubiquitin interaction remains elusive. In the present study, replica exchange molecular dynamics simulations were performed to examine the conformational dynamics of Parkin in monomer and phospho-ubiquitin-bound states. In the Parkin monomer state, high structural flexibilities were observed in the majority of regions of Parkin particularly in the loop domain between the ubiquitin-like (UBL) and really interesting new gene (RING)0 domain. Binding of phospho-ubiquitin stabilizes the RING1/RING in between RING interface but destabilizes the RING1-UBL interface. Furthermore, using steered molecular dynamics simulations of Parkin mutations, it was demonstrated that salt bridge interactions contribute significantly to the interdomain interactions between the RING1 and UBL domain. Taken together, the results of the present study revealed the conformational dynamics of human full-length Parkin in monomer and phospho-ubiquitin-bound states, providing insights into designing potential therapeutics against Parkinson's disease. PMID:28765939

Validating the disruption of proliferating cell nuclear antigen interactions in the development of targeted cancer therapeutics.

PubMed

Smith, Shanna J; Hickey, Robert J; Malkas, Linda H

2016-01-01

Human DNA replication and repair is a highly coordinated process involving the specifically timed actions of numerous proteins and enzymes. Many of these proteins require interaction with proliferating cell nuclear antigen (PCNA) for activation within the process. The interdomain connector loop (IDCL) of PCNA provides a docking site for many of those proteins, suggesting that this region is critically important in the regulation of cellular function. Previous work in this laboratory has demonstrated that a peptide mimicking a specific region of the IDCL (caPeptide) has the ability to disrupt key protein-protein interactions between PCNA and its binding partners, thereby inhibiting DNA replication within the cells. In this study, we confirm the ability of the caPeptide to disrupt DNA replication function using both intact cell and in vitro DNA replication assays. Further, we were able to demonstrate that treatment with caPeptide results in a decrease of polymerase δ activity that correlates with the observed decrease in DNA replication. We have also successfully developed a surface plasmon resonance (SPR) assay to validate the disruption of the PCNA-pol δ interaction with caPeptide.

A Bacterial Quorum-Sensing Precursor Induces Mortality in the Marine Coccolithophore, Emiliania huxleyi.

PubMed

Harvey, Elizabeth L; Deering, Robert W; Rowley, David C; El Gamal, Abrahim; Schorn, Michelle; Moore, Bradley S; Johnson, Matthew D; Mincer, Tracy J; Whalen, Kristen E

2016-01-01

Interactions between phytoplankton and bacteria play a central role in mediating biogeochemical cycling and food web structure in the ocean. However, deciphering the chemical drivers of these interspecies interactions remains challenging. Here, we report the isolation of 2-heptyl-4-quinolone (HHQ), released by Pseudoalteromonas piscicida, a marine gamma-proteobacteria previously reported to induce phytoplankton mortality through a hitherto unknown algicidal mechanism. HHQ functions as both an antibiotic and a bacterial signaling molecule in cell-cell communication in clinical infection models. Co-culture of the bloom-forming coccolithophore, Emiliania huxleyi with both live P. piscicida and cell-free filtrates caused a significant decrease in algal growth. Investigations of the P. piscicida exometabolome revealed HHQ, at nanomolar concentrations, induced mortality in three strains of E. huxleyi. Mortality of E. huxleyi in response to HHQ occurred slowly, implying static growth rather than a singular loss event (e.g., rapid cell lysis). In contrast, the marine chlorophyte, Dunaliella tertiolecta and diatom, Phaeodactylum tricornutum were unaffected by HHQ exposures. These results suggest that HHQ mediates the type of inter-domain interactions that cause shifts in phytoplankton population dynamics. These chemically mediated interactions, and other like it, ultimately influence large-scale oceanographic processes.

Probing Gαi1 Protein Activation at Single Amino Acid Resolution

PubMed Central

Sun, Dawei; Maeda, Shoji; Matkovic, Milos; Mendieta, Sandro; Mayer, Daniel; Dawson, Roger; Schertler, Gebhard F.X.; Madan Babu, M.; Veprintsev, Dmitry B.

2016-01-01

We present comprehensive single amino acid resolution maps of the residues stabilising the human Gαi1 subunit in nucleotide- and receptor-bound states. We generated these maps by measuring the effects of alanine mutations on the stability of Gαi1 and of the rhodopsin-Gαi1 complex. We identified stabilization clusters in the GTPase and helical domains responsible for structural integrity and the conformational changes associated with activation. In activation cluster I, helices α1 and α5 pack against strands β1-3 to stabilize the nucleotide-bound states. In the receptor-bound state, these interactions are replaced by interactions between α5 and strands β4-6. Key residues in this cluster are Y320, crucial for the stabilization of the receptor-bound state, and F336, which stabilizes nucleotide-bound states. Destabilization of helix α1, caused by rearrangement of this activation cluster, leads to the weakening of the inter-domain interface and release of GDP. PMID:26258638

Functional map of arrestin binding to phosphorylated opsin, with and without agonist.

PubMed

Peterhans, Christian; Lally, Ciara C M; Ostermaier, Martin K; Sommer, Martha E; Standfuss, Jörg

2016-06-28

Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct.

The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs.

PubMed

Sunita, S; Schwartz, Samantha L; Conn, Graeme L

2015-11-20

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain and a C-terminal kinase domain interposed by an unstructured ∼80-residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR) and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries and that each exhibits comparable polyinosinic-cytidylic (poly(rI:rC)) dsRNA activation profiles and binding affinities for adenoviral virus-associated RNA I (VA RNAI) and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain-swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rat PKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism of allosteric propagation across a β-sheet structure investigated by molecular dynamics simulations.

PubMed

Interlandi, Gianluca; Thomas, Wendy E

2016-07-01

The bacterial adhesin FimH consists of an allosterically regulated mannose-binding lectin domain and a covalently linked inhibitory pilin domain. Under normal conditions, the two domains are bound to each other, and FimH interacts weakly with mannose. However, under tensile force, the domains separate and the lectin domain undergoes conformational changes that strengthen its bond with mannose. Comparison of the crystallographic structures of the low and the high affinity state of the lectin domain reveals conformational changes mainly in the regulatory inter-domain region, the mannose binding site and a large β sheet that connects the two distally located regions. Here, molecular dynamics simulations investigated how conformational changes are propagated within and between different regions of the lectin domain. It was found that the inter-domain region moves towards the high affinity conformation as it becomes more compact and buries exposed hydrophobic surface after separation of the pilin domain. The mannose binding site was more rigid in the high affinity state, which prevented water penetration into the pocket. The large central β sheet demonstrated a soft spring-like twisting. Its twisting motion was moderately correlated to fluctuations in both the regulatory and the binding region, whereas a weak correlation was seen in a direct comparison of these two distal sites. The results suggest a so called "population shift" model whereby binding of the lectin domain to either the pilin domain or mannose locks the β sheet in a rather twisted or flat conformation, stabilizing the low or the high affinity state, respectively. Proteins 2016; 84:990-1008. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Mechanical coupling in myosin V: a simulation study

PubMed Central

Ovchinnikov, Victor; Trout, Bernhardt L.

2009-01-01

Myosin motor function depends on the interaction between different domains that transmit information from one part of the molecule to another. The inter-domain coupling in myosin V is studied with Restrained Targeted Molecular Dynamics (RTMD) using an all-atom representation in explicit solvent. To elucidate the origin of the conformational change due to the binding of ATP, targeting forces are applied to small sets of atoms (the forcing sets, FS) in the direction of their displacement from the rigor conformation, which has a closed actin-binding cleft, to the post-rigor conformation, in which the cleft is open. The ‘minimal’ FS that results in extensive structural changes in the overall myosin conformation is comprised of the ATP, Switch 1, and the nearby HF, HG and HH helices. Addition of switch 2 to the forcing set is required to achieve a complete opening of the actin-binding cleft. The RTMD simulations reveal the mechanical coupling pathways between (i) the nucleotide-binding pocket (NBP) and the actin-binding cleft, (ii) the NBP and the converter, and (iii) the actin-binding cleft and the converter. Closing of the NBP due to ATP binding is tightly coupled to the opening of the cleft, and leads to the rupture of a key hydrogen bond (F441N/A684O) between switch 2 and the SH1 helix. The actin-binding cleft may mediate the rupture of this bond via a connection between the HW helix, the Relay helix, and Switch 2. The findings are consistent with experimental studies and a recent normal mode analysis. The present method is expected to be useful more generally in studies of inter-domain coupling in proteins. PMID:19853615

Mechanical coupling in myosin V: a simulation study.

PubMed

Ovchinnikov, Victor; Trout, Bernhardt L; Karplus, Martin

2010-01-29

Myosin motor function depends on the interaction between different domains that transmit information from one part of the molecule to another. The interdomain coupling in myosin V is studied with restrained targeted molecular dynamics using an all-atom representation in explicit solvent. To elucidate the origin of the conformational change due to the binding of ATP, targeting forces are applied to small sets of atoms (the forcing sets, FSs) in the direction of their displacement from the rigor conformation, which has a closed actin-binding cleft, to the post-rigor conformation, in which the cleft is open. The "minimal" FS that results in extensive structural changes in the overall myosin conformation is composed of ATP, switch 1, and the nearby HF, HG, and HH helices. Addition of switch 2 to the FS is required to achieve a complete opening of the actin-binding cleft. The restrained targeted molecular dynamics simulations reveal the mechanical coupling pathways between (i) the nucleotide-binding pocket (NBP) and the actin-binding cleft, (ii) the NBP and the converter, and (iii) the actin-binding cleft and the converter. Closing of the NBP due to ATP binding is tightly coupled to the opening of the cleft and leads to the rupture of a key hydrogen bond (F441N/A684O) between switch 2 and the SH1 helix. The actin-binding cleft may mediate the rupture of this bond via a connection between the HW helix, the relay helix, and switch 2. The findings are consistent with experimental studies and a recent normal mode analysis. The present method is expected to be useful more generally in studies of interdomain coupling in proteins.

Construction of Recombinant Hemagglutinin Derived from the Gingipain-Encoding Gene of Porphyromonas gingivalis, Identification of Its Target Protein on Erythrocytes, and Inhibition of Hemagglutination by an Interdomain Regional Peptide▿

PubMed Central

Sakai, Eiko; Naito, Mariko; Sato, Keiko; Hotokezaka, Hitoshi; Kadowaki, Tomoko; Kamaguchi, Arihide; Yamamoto, Kenji; Okamoto, Kuniaki; Nakayama, Koji

2007-01-01

Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44720-1081, a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44720-1138, did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44720-1138 but not in HGP44720-1081, could bind HGP44720-1081 in a dose-dependent manner and effectively inhibited HGP44720-1081-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44720-1081-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44720-1081-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44720-1081 could bind to asialoglycophorin with a dissociation constant of 3.0 × 10−7 M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A. PMID:17384191

Polyglycine hydrolases secreted by pathogenic fungi

USDA-ARS?s Scientific Manuscript database

Pathogens are known to produce proteases that target host defense proteins. Here we describe polyglycine hydrolases, fungal proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine interdomain linker of targeted plant defense chitinases. Polyglycine hydrolases were puri...

A whole genome analysis reveals the presence of a plant PR1 sequence in the potato pathogen Streptomyces scabies and other Streptomyces species.

PubMed

Armijos-Jaramillo, Vinicio; Santander-Gordón, Daniela; Soria, Rosa; Pazmiño-Betancourth, Mauro; Echeverría, María Cristina

2017-09-01

Streptomyces scabies is a common soil bacterium that causes scab symptoms in potatoes. Strong evidence indicates horizontal gene transfer (HGT) among bacteria has influenced the evolution of this plant pathogen and other Streptomyces spp. To extend the study of the HGT to the Streptomyces genus, we explored the effects of the inter-domain HGT in the S. scabies genome. We employed a semi-automatic pipeline based on BLASTp searches and phylogenetic reconstruction. The data show low impact of inter-domain HGT in the S. scabies genome; however, we found a putative plant pathogenesis related 1 (PR1) sequence in the genome of S. scabies and other species of the genus. It is possible that this gene could be used by S. scabies to out-compete other soil organisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Integrins protect cardiomyocytes from ischemia/reperfusion injury

PubMed Central

Okada, Hideshi; Lai, N. Chin; Kawaraguchi, Yoshitaka; Liao, Peter; Copps, Jeffrey; Sugano, Yasuo; Okada-Maeda, Sunaho; Banerjee, Indroneal; Schilling, Jan M.; Gingras, Alexandre R.; Asfaw, Elizabeth K.; Suarez, Jorge; Kang, Seok-Min; Perkins, Guy A.; Au, Carol G.; Israeli-Rosenberg, Sharon; Manso, Ana Maria; Liu, Zheng; Milner, Derek J.; Kaufman, Stephen J.; Patel, Hemal H.; Roth, David M.; Hammond, H. Kirk; Taylor, Susan S.; Dillmann, Wolfgang H.; Goldhaber, Joshua I.; Ross, Robert S.

2013-01-01

Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165–175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury. PMID:24091324

A Traveling Wave Ion Mobility Spectrometry (TWIMS) Study of the Robo1-Heparan Sulfate Interaction

NASA Astrophysics Data System (ADS)

Zhao, Yuejie; Yang, Jeong Yeh; Thieker, David F.; Xu, Yongmei; Zong, Chengli; Boons, Geert-Jan; Liu, Jian; Woods, Robert J.; Moremen, Kelley W.; Amster, I. Jonathan

2018-03-01

Roundabout 1 (Robo1) interacts with its receptor Slit to regulate axon guidance, axon branching, and dendritic development in the nervous system and to regulate morphogenesis and many cell functions in the nonneuronal tissues. This interaction is known to be critically regulated by heparan sulfate (HS). Previous studies suggest that HS is required to promote the binding of Robo1 to Slit to form the minimal signaling complex, but the molecular details and the structural requirements of HS for this interaction are still unclear. Here, we describe the application of traveling wave ion mobility spectrometry (TWIMS) to study the conformational details of the Robo1-HS interaction. The results suggest that Robo1 exists in two conformations that differ by their compactness and capability to interact with HS. The results also suggest that the highly flexible interdomain hinge region connecting the Ig1 and Ig2 domains of Robo1 plays an important functional role in promoting the Robo1-Slit interaction. Moreover, variations in the sulfation pattern and size of HS were found to affect its binding affinity and selectivity to interact with different conformations of Robo1. Both MS measurements and CIU experiments show that the Robo1-HS interaction requires the presence of a specific size and pattern of modification of HS. Furthermore, the effect of N-glycosylation on the conformation of Robo1 and its binding modes with HS is reported. [Figure not available: see fulltext.

Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

PubMed

Shem-Ad, Tzilhav; Irit, Orr; Yifrach, Ofer

2013-01-01

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

Inhibition of polygylcine hydrolases by substrate analog peptides

USDA-ARS?s Scientific Manuscript database

Polyglycine hydrolases are proteases secreted by fungal pathogens that target corn defense chitinases. They cleave interdomain glycine-glycine bonds within a polyglycine linker, separating substrate chitinases into two single domain proteins. Polyglycine hydrolases consist of 640 amino acids with a ...

DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a

PubMed Central

Walters, Kylie J.; Lech, Patrycja J.; Goh, Amanda M.; Wang, Qinghua; Howley, Peter M.

2003-01-01

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation. PMID:14557549

Actin-induced dimerization of palladin promotes actin-bundling

PubMed Central

Vattepu, Ravi; Yadav, Rahul; Beck, Moriah R

2015-01-01

A subset of actin binding proteins is able to form crosslinks between two or more actin filaments, thus producing structures of parallel or networked bundles. These actin crosslinking proteins interact with actin through either bivalent binding or dimerization. We recently identified two binding sites within the actin binding domain of palladin, an actin crosslinking protein that plays an important role in normal cell adhesion and motility during wound healing and embryonic development. In this study, we show that actin induces dimerization of palladin. Furthermore, the extent of dimerization reflects earlier comparisons of actin binding and bundling between different domains of palladin. On the basis of these results we hypothesized that actin binding may promote a conformational change that results in dimerization of palladin, which in turn may drive the crosslinking of actin filaments. The proximal distance between two actin binding sites on crosslinking proteins determines the ultrastructural properties of the filament network, therefore we also explored interdomain interactions using a combination of chemical crosslinking experiments and actin cosedimentation assays. Limited proteolysis data reveals that palladin is less susceptible to enzyme digestion after actin binding. Our results suggest that domain movements in palladin are necessary for interactions with actin and are induced by interactions with actin filaments. Accordingly, we put forth a model linking the structural changes to functional dynamics. PMID:25307943

Molecular dynamics simulations of human E3 ubiquitin ligase Parkin.

PubMed

Qiu, Shi; Zhu, Shun; Xu, Shan; Han, Yanyan; Liu, Wen; Zuo, Ji

2017-10-01

Human E3 ubiquitin protein ligase parkin (Parkin) mediates mitophagy to maintain mitochondrial homeostasis. Parkin mutations are common genetic causes of early onset familial Parkinson's disease. The molecular mechanism of Parkin activation has been widely studied with emerging evidence suggesting an essential role of the phosphorylated (phospho)‑ubiquitin interaction. However, the underlying mecha-nism of the phospho‑ubiquitin interaction remains elusive. In the present study, replica exchange molecular dynamics simulations were performed to examine the conformational dynamics of Parkin in monomer and phospho‑ubiquitin‑bound states. In the Parkin monomer state, high structural flexi-bilities were observed in the majority of regions of Parkin particularly in the loop domain between the ubiquitin‑like (UBL) and really interesting new gene (RING)0 domain. Binding of phospho‑ubiquitin stabilizes the RING1/RING in between RING interface but destabilizes the RING1‑UBL interface. Furthermore, using steered molecular dynamics simulations of Parkin mutations, it was demonstrated that salt bridge interactions contribute significantly to the interdomain interactions between the RING1 and UBL domain. Taken together, the results of the present study revealed the conformational dynamics of human full‑length Parkin in monomer and phospho‑ubiquitin‑bound states, providing insights into designing potential therapeutics against Parkinson's disease.

Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1

PubMed Central

Iwig, Jeffrey S; Vercoulen, Yvonne; Das, Rahul; Barros, Tiago; Limnander, Andre; Che, Yan; Pelton, Jeffrey G; Wemmer, David E; Roose, Jeroen P; Kuriyan, John

2013-01-01

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals. DOI: http://dx.doi.org/10.7554/eLife.00813.001 PMID:23908768

Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I

NASA Astrophysics Data System (ADS)

Venditti, Vincenzo; Tugarinov, Vitali; Schwieters, Charles D.; Grishaev, Alexander; Clore, G. Marius

2015-01-01

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate and the inhibitor α-ketoglutarate, on the structure and dynamics of EI using NMR, small-angle X-ray scattering and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

PubMed

Karow, Anne R; Theissen, Bettina; Klostermeier, Dagmar

2007-01-01

RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

Recognition and cleavage of corn defense chitinases by fungal polyglycine hydrolases

USDA-ARS?s Scientific Manuscript database

Polyglycine hydrolases are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn. Polyglycine hydrolases are novel proteins in terms of activity and sequence. The objective of the study is to und...

Recognition of corn defense chitinases by fungal polyglycine hydrolases

USDA-ARS?s Scientific Manuscript database

Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a specific pe...

Role Conflict and Faculty Life Satisfaction.

ERIC Educational Resources Information Center

Olsen, Deborah; Near, Janet P.

1994-01-01

A study of research university faculty in first (n=52) and third (n=47) years of appointment investigated relationships among work and nonwork satisfaction, interdomain conflict, and life satisfaction. Findings indicated that balance and conflict explained variance in life satisfaction beyond that explained by job and nonwork satisfaction. Changes…

CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions

PubMed Central

Dai, Gucan

2013-01-01

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the L633P mutation. PMID:23552282

CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions.

PubMed

Dai, Gucan; Varnum, Michael D

2013-07-15

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the L633P mutation.

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.

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET.

PubMed

Sikor, Martin; Mapa, Koyeli; von Voithenberg, Lena Voith; Mokranjac, Dejana; Lamb, Don C

2013-05-29

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.

Slow domain reconfiguration causes power-law kinetics in a two-state enzyme.

PubMed

Grossman-Haham, Iris; Rosenblum, Gabriel; Namani, Trishool; Hofmann, Hagen

2018-01-16

Protein dynamics are typically captured well by rate equations that predict exponential decays for two-state reactions. Here, we describe a remarkable exception. The electron-transfer enzyme quiescin sulfhydryl oxidase (QSOX), a natural fusion of two functionally distinct domains, switches between open- and closed-domain arrangements with apparent power-law kinetics. Using single-molecule FRET experiments on time scales from nanoseconds to milliseconds, we show that the unusual open-close kinetics results from slow sampling of an ensemble of disordered domain orientations. While substrate accelerates the kinetics, thus suggesting a substrate-induced switch to an alternative free energy landscape of the enzyme, the power-law behavior is also preserved upon electron load. Our results show that the slow sampling of open conformers is caused by a variety of interdomain interactions that imply a rugged free energy landscape, thus providing a generic mechanism for dynamic disorder in multidomain enzymes.

Polyglycine hydrolases: fungal b-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases

USDA-ARS?s Scientific Manuscript database

Polyglycine hydrolases are secreted fungal proteases that cleave glycine-glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochli...

Geo-Based Inter-Domain Routing (GIDR) Protocol for MANETS

DTIC Science & Technology

2009-10-01

routing, and support for node mobility. Crowcroft et al. proposed Plutarch as architecture to translate address spaces and transport protocols among...Warfield, “ Plutarch : an argument for network pluralism,” ACM Computer Communication Review, vol. 33, no. 4, pp. 258–266, 2003. [6] S. Schmid, L

Minimizing Wide-Area Performance Disruptions in Inter-Domain Routing

DTIC Science & Technology

2011-09-01

Servers As another example, we saw the average round-trip time double for an ISP in Malaysia . The RTT increase was caused by a traffic shift to different... censorship , conduct wiretapping, or offer poor performance. This is achieved by applying regular expressions to the AS-PATH to assign lower preference

Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: Photoconversion and signal transduction

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

Yang, Xiaojing; Kuk, Jane; Moffat, Keith

2008-11-12

Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria via reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here we report the crystal structure at 2.9 {angstrom} resolution of a bacteriophytochrome from Pseudomonas aeruginosa with an intact, fully photoactive photosensory core domain in its dark-adapted Pfr state. This structure reveals how unusual interdomain interactions, including a knot and an 'arm' structure near the chromophore site, bring together the PAS (Per-ARNT-Sim), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA), and PHY (phytochrome) domains to achieve Pr/Pfr photoconversion. The PAS, GAF, and PHY domains have topologic elements in common andmore » may have a single evolutionary origin. We identify key interactions that stabilize the chromophore in the Pfr state and provide structural and mutational evidence to support the essential role of the PHY domain in efficient Pr/Pfr photoconversion. We also identify a pair of conserved residues that may undergo concerted conformational changes during photoconversion. Modeling of the full-length bacteriophytochrome structure, including its output histidine kinase domain, suggests how local structural changes originating in the photosensory domain modulate interactions between long, cross-domain signaling helices at the dimer interface and are transmitted to the spatially distant effector domain, thereby regulating its histidine kinase activity.« less

Electrostatic Interactions as Mediators in the Allosteric Activation of Protein Kinase A RIα.

PubMed

P Barros, Emília; Malmstrom, Robert D; Nourbakhsh, Kimya; Del Rio, Jason C; Kornev, Alexandr P; Taylor, Susan S; Amaro, Rommie E

2017-03-14

Close-range electrostatic interactions that form salt bridges are key components of protein stability. Here we investigate the role of these charged interactions in modulating the allosteric activation of protein kinase A (PKA) via computational and experimental mutational studies of a conserved basic patch located in the regulatory subunit's B/C helix. Molecular dynamics simulations evidenced the presence of an extended network of fluctuating salt bridges spanning the helix and connecting the two cAMP binding domains in its extremities. Distinct changes in the flexibility and conformational free energy landscape induced by the separate mutations of Arg239 and Arg241 suggested alteration of cAMP-induced allosteric activation and were verified through in vitro fluorescence polarization assays. These observations suggest a mechanical aspect to the allosteric transition of PKA, with Arg239 and Arg241 acting in competition to promote the transition between the two protein functional states. The simulations also provide a molecular explanation for the essential role of Arg241 in allowing cooperative activation, by evidencing the existence of a stable interdomain salt bridge with Asp267. Our integrated approach points to the role of salt bridges not only in protein stability but also in promoting conformational transition and function.

Establishing a Role for Bacterial Cellulose in Environmental Interactions: Lessons Learned from Diverse Biofilm-Producing Proteobacteria

PubMed Central

Augimeri, Richard V.; Varley, Andrew J.; Strap, Janice L.

2015-01-01

Bacterial cellulose (BC) serves as a molecular glue to facilitate intra- and inter-domain interactions in nature. Biosynthesis of BC-containing biofilms occurs in a variety of Proteobacteria that inhabit diverse ecological niches. The enzymatic and regulatory systems responsible for the polymerization, exportation, and regulation of BC are equally as diverse. Though the magnitude and environmental consequences of BC production are species-specific, the common role of BC-containing biofilms is to establish close contact with a preferred host to facilitate efficient host–bacteria interactions. Universally, BC aids in attachment, adherence, and subsequent colonization of a substrate. Bi-directional interactions influence host physiology, bacterial physiology, and regulation of BC biosynthesis, primarily through modulation of intracellular bis-(3′→5′)-cyclic diguanylate (c-di-GMP) levels. Depending on the circumstance, BC producers exhibit a pathogenic or symbiotic relationship with plant, animal, or fungal hosts. Rhizobiaceae species colonize plant roots, Pseudomonadaceae inhabit the phyllosphere, Acetobacteriaceae associate with sugar-loving insects and inhabit the carposphere, Enterobacteriaceae use fresh produce as vehicles to infect animal hosts, and Vibrionaceae, particularly Aliivibrio fischeri, colonize the light organ of squid. This review will highlight the diversity of the biosynthesis and regulation of BC in nature by discussing various examples of Proteobacteria that use BC-containing biofilms to facilitate host–bacteria interactions. Through discussion of current data we will establish new directions for the elucidation of BC biosynthesis, its regulation and its ecophysiological roles. PMID:26635751

Crystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design.

PubMed

Corradi, Hazel R; Schwager, Sylva L U; Nchinda, Aloysius T; Sturrock, Edward D; Acharya, K Ravi

2006-03-31

Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors.

Structural basis of arrestin-3 activation and signaling

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

Chen, Qiuyan; Perry, Nicole A.; Vishnivetskiy, Sergey A.

A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP6) is a non-receptor activator of arrestin-3 and report the structure of IP6-activated arrestin-3 at 2.4-Å resolution. IP6-activated arrestin-3 exhibits an inter-domain twist and a displaced C-tail, hallmarks of active arrestin. IP6 binds to the arrestin phosphate sensor, and is stabilized by trimerization. Analysis of the trimerization surface, which is also the receptor-binding surface, suggests a feature called the finger loop as a key region of the activation sensor. We show that finger loop helicity and flexibility may underliemore » coupling to hundreds of diverse receptors and also promote arrestin-3 activation by IP6. Importantly, we show that effector-binding sites on arrestins have distinct conformations in the basal and activated states, acting as switch regions. These switch regions may work with the inter-domain twist to initiate and direct arrestin-mediated signaling.« less

Temperature dependence of internal friction in enzyme reactions.

PubMed

Rauscher, Anna Á; Simon, Zoltán; Szöllosi, Gergely J; Gráf, László; Derényi, Imre; Malnasi-Csizmadia, Andras

2011-08-01

Our aim was to elucidate the physical background of internal friction of enzyme reactions by investigating the temperature dependence of internal viscosity. By rapid transient kinetic methods, we directly measured the rate constant of trypsin 4 activation, which is an interdomain conformational rearrangement, as a function of temperature and solvent viscosity. We found that the apparent internal viscosity shows an Arrhenius-like temperature dependence, which can be characterized by the activation energy of internal friction. Glycine and alanine mutations were introduced at a single position of the hinge of the interdomain region to evaluate how the flexibility of the hinge affects internal friction. We found that the apparent activation energies of the conformational change and the internal friction are interconvertible parameters depending on the protein flexibility. The more flexible a protein was, the greater proportion of the total activation energy of the reaction was observed as the apparent activation energy of internal friction. Based on the coupling of the internal and external movements of the protein during its conformational change, we constructed a model that quantitatively relates activation energy, internal friction, and protein flexibility.

Probing Molecular Mechanisms of the Hsp90 Chaperone: Biophysical Modeling Identifies Key Regulators of Functional Dynamics

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2012-01-01

Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients. PMID:22624053

Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

PubMed Central

Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

2012-01-01

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

Functional Domains of the TOL Plasmid Transcription Factor XylS

PubMed Central

Kaldalu, Niilo; Toots, Urve; de Lorenzo, Victor; Ustav, Mart

2000-01-01

The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions. PMID:10648539

Interface Matters: The Stiffness Route to Stability of a Thermophilic Tetrameric Malate Dehydrogenase

PubMed Central

Kalimeri, Maria; Girard, Eric; Madern, Dominique; Sterpone, Fabio

2014-01-01

In this work we investigate by computational means the behavior of two orthologous bacterial proteins, a mesophilic and a thermophilic tetrameric malate dehydrogenase (MalDH), at different temperatures. Namely, we quantify how protein mechanical rigidity at different length- and time-scales correlates to protein thermophilicity as commonly believed. In particular by using a clustering analysis strategy to explore the conformational space of the folded proteins, we show that at ambient conditions and at the molecular length-scale the thermophilic variant is indeed more rigid that the mesophilic one. This rigidification is the result of more efficient inter-domain interactions, the strength of which is further quantified via ad hoc free energy calculations. When considered isolated, the thermophilic domain is indeed more flexible than the respective mesophilic one. Upon oligomerization, the induced stiffening of the thermophilic protein propagates from the interface to the active site where the loop, controlling the access to the catalytic pocket, anchors down via an extended network of ion-pairs. On the contrary in the mesophilic tetramer the loop is highly mobile. Simulations at high temperature, could not re-activate the mobility of the loop in the thermophile. This finding opens questions on the similarities of the binding processes for these two homologues at their optimal working temperature and suggests for the thermophilic variant a possible cooperative role of cofactor/substrate. PMID:25437494

Interface matters: the stiffness route to stability of a thermophilic tetrameric malate dehydrogenase.

PubMed

Kalimeri, Maria; Girard, Eric; Madern, Dominique; Sterpone, Fabio

2014-01-01

In this work we investigate by computational means the behavior of two orthologous bacterial proteins, a mesophilic and a thermophilic tetrameric malate dehydrogenase (MalDH), at different temperatures. Namely, we quantify how protein mechanical rigidity at different length- and time-scales correlates to protein thermophilicity as commonly believed. In particular by using a clustering analysis strategy to explore the conformational space of the folded proteins, we show that at ambient conditions and at the molecular length-scale the thermophilic variant is indeed more rigid that the mesophilic one. This rigidification is the result of more efficient inter-domain interactions, the strength of which is further quantified via ad hoc free energy calculations. When considered isolated, the thermophilic domain is indeed more flexible than the respective mesophilic one. Upon oligomerization, the induced stiffening of the thermophilic protein propagates from the interface to the active site where the loop, controlling the access to the catalytic pocket, anchors down via an extended network of ion-pairs. On the contrary in the mesophilic tetramer the loop is highly mobile. Simulations at high temperature, could not re-activate the mobility of the loop in the thermophile. This finding opens questions on the similarities of the binding processes for these two homologues at their optimal working temperature and suggests for the thermophilic variant a possible cooperative role of cofactor/substrate.

The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

PubMed Central

Shamayeva, Katsiaryna; Guzanova, Alena; Řeha, David; Csefalvay, Eva; Carey, Jannette; Weiserova, Marie

2017-01-01

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested. PMID:28133570

Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

PubMed Central

Lin, Hong-En; Tsai, Wen-Yang; Liu, I-Ju; Li, Pi-Chun; Liao, Mei-Ying; Tsai, Jih-Jin; Wu, Yi-Chieh; Lai, Chih-Yun; Lu, Chih-Hsuan; Huang, Jyh-Hsiung; Chang, Gwong-Jen; Wu, Han-Chung; Wang, Wei-Kung

2012-01-01

Background The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. PMID:22235356

Domain Motion Enhanced (DoME) Model for Efficient Conformational Sampling of Multidomain Proteins.

PubMed

Kobayashi, Chigusa; Matsunaga, Yasuhiro; Koike, Ryotaro; Ota, Motonori; Sugita, Yuji

2015-11-19

Large conformational changes of multidomain proteins are difficult to simulate using all-atom molecular dynamics (MD) due to the slow time scale. We show that a simple modification of the structure-based coarse-grained (CG) model enables a stable and efficient MD simulation of those proteins. "Motion Tree", a tree diagram that describes conformational changes between two structures in a protein, provides information on rigid structural units (domains) and the magnitudes of domain motions. In our new CG model, which we call the DoME (domain motion enhanced) model, interdomain interactions are defined as being inversely proportional to the magnitude of the domain motions in the diagram, whereas intradomain interactions are kept constant. We applied the DoME model in combination with the Go model to simulations of adenylate kinase (AdK). The results of the DoME-Go simulation are consistent with an all-atom MD simulation for 10 μs as well as known experimental data. Unlike the conventional Go model, the DoME-Go model yields stable simulation trajectories against temperature changes and conformational transitions are easily sampled despite domain rigidity. Evidently, identification of domains and their interfaces is useful approach for CG modeling of multidomain proteins.

Temperature-accelerated molecular dynamics gives insights into globular conformations sampled in the free state of the AC catalytic domain.

PubMed

Selwa, Edithe; Huynh, Tru; Ciccotti, Giovanni; Maragliano, Luca; Malliavin, Thérèse E

2014-10-01

The catalytic domain of the adenyl cyclase (AC) toxin from Bordetella pertussis is activated by interaction with calmodulin (CaM), resulting in cAMP overproduction in the infected cell. In the X-ray crystallographic structure of the complex between AC and the C terminal lobe of CaM, the toxin displays a markedly elongated shape. As for the structure of the isolated protein, experimental results support the hypothesis that more globular conformations are sampled, but information at atomic resolution is still lacking. Here, we use temperature-accelerated molecular dynamics (TAMD) simulations to generate putative all-atom models of globular conformations sampled by CaM-free AC. As collective variables, we use centers of mass coordinates of groups of residues selected from the analysis of standard molecular dynamics (MD) simulations. Results show that TAMD allows extended conformational sampling and generates AC conformations that are more globular than in the complexed state. These structures are then refined via energy minimization and further unrestrained MD simulations to optimize inter-domain packing interactions, thus resulting in the identification of a set of hydrogen bonds present in the globular conformations. © 2014 Wiley Periodicals, Inc.

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1.

PubMed

Prattes, Michael; Loibl, Mathias; Zisser, Gertrude; Luschnig, Daniel; Kappel, Lisa; Rössler, Ingrid; Grassegger, Manuela; Hromic, Altijana; Krieger, Elmar; Gruber, Karl; Pertschy, Brigitte; Bergler, Helmut

2017-03-17

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.

Molecular mechanism of a COOH-terminal gating determinant in the ROMK channel revealed by a Bartter's disease mutation

PubMed Central

Flagg, Thomas P; Yoo, Dana; Sciortino, Christopher M; Tate, Margaret; Romero, Michael F; Welling, Paul A

2002-01-01

The ROMK subtypes of inward-rectifier K+ channels mediate potassium secretion and regulate NaCl reabsorption in the kidney. Loss-of-function mutations in this pH-sensitive K+ channel cause Bartter's disease, a familial salt wasting nephropathy. One disease-causing mutation truncates the extreme COOH-terminus and induces a closed gating conformation. Here we identify a region within the deleted domain that plays an important role in pH-dependent gating. The domain contains a structural element that functionally interacts with the pH sensor in the cytoplasmic NH2-terminus to set a physiological range of pH sensitivity. Removal of the domain shifts the pKa towards alkaline pH values, causing channel inactivation under physiological conditions. Suppressor mutations within the pH sensor rescued channel gating and trans addition of the cognate peptide restored pH sensitivity. A specific interdomain interaction was revealed in an in vitro protein-protein binding assay between the NH2- and COOH-terminal cytoplasmic domains expressed as bacterial fusion proteins. These results provide new insights into the molecular mechanisms underlying Kir channel regulation and channel gating defects that are associated with Bartter's disease. PMID:12381810

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

Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonicalmore » PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

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

Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo

Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain amore » canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2015-01-01

Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones.

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins

PubMed Central

Stetz, Gabrielle; Verkhivker, Gennady M.

2015-01-01

Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones. PMID:26619280

Cysteine residues in the nucleotide binding domains regulate the conductance state of CFTR channels.

PubMed Central

Harrington, Melissa A; Kopito, Ron R

2002-01-01

Gating of cystic fibrosis transmembrane conductance regulator (CFTR) channels requires intermolecular or interdomain interactions, but the exact nature and physiological significance of those interactions remains uncertain. Subconductance states of the channel may result from alterations in interactions among domains, and studying mutant channels enriched for a single conductance type may elucidate those interactions. Analysis of CFTR channels in inside-out patches revealed that mutation of cysteine residues in NBD1 and NBD2 affects the frequency of channel opening to the full-size versus a 3-pS subconductance. Mutating cysteines in NBD1 resulted in channels that open almost exclusively to the 3-pS subconductance, while mutations of cysteines in NBD2 decreased the frequency of subconductance openings. Wild-type channels open to both size conductances and make fast transitions between them within a single open burst. Full-size and subconductance openings of both mutant and wild-type channels are similarly activated by ATP and phosphorylation. However, the different size conductances open very differently in the presence of a nonhydrolyzable ATP analog, with subconductance openings significantly shortened by ATPgammaS, while full-size channels are locked open. In wild-type channels, reducing conditions increase the frequency and decrease the open time of subconductance channels, while oxidizing conditions decrease the frequency of subconductance openings. In contrast, in the cysteine mutants studied, altering redox potential has little effect on gating of the subconductance. PMID:11867445

Structure and Functional Characterization of the RNA-Binding Element of the NLRX1 Innate Immune Modulator

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

Hong, Minsun; Yoon, Sung-il; Wilson, Ian A.

2012-06-20

Mitochondrial NLRX1 is a member of the family of nucleotide-binding domain and leucine-rich-repeat-containing proteins (NLRs) that mediate host innate immunity as intracellular surveillance sensors against common molecular patterns of invading pathogens. NLRX1 functions in antiviral immunity, but the molecular mechanism of its ligand-induced activation is largely unknown. The crystal structure of the C-terminal fragment (residues 629975) of human NLRX1 (cNLRX1) at 2.65 {angstrom} resolution reveals that cNLRX1 consists of an N-terminal helical (LRRNT) domain, central leucine-rich repeat modules (LRRM), and a C-terminal three-helix bundle (LRRCT). cNLRX1 assembles into a compact hexameric architecture that is stabilized by intersubunit and interdomain interactionsmore » of LRRNT and LRRCT in the trimer and dimer components of the hexamer, respectively. Furthermore, we find that cNLRX1 interacts directly with RNA and supports a role for NLRX1 in recognition of intracellular viral RNA in antiviral immunity.« less

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET

PubMed Central

Sikor, Martin; Mapa, Koyeli; von Voithenberg, Lena Voith; Mokranjac, Dejana; Lamb, Don C

2013-01-01

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions. PMID:23624933

Structure-guided mutagenesis reveals a hierarchical mechanism of Parkin activation

PubMed Central

Tang, Matthew Y.; Vranas, Marta; Krahn, Andrea I.; Pundlik, Shayal; Trempe, Jean- François; Fon, Edward A.

2017-01-01

Parkin and PINK1 function in a common pathway to clear damaged mitochondria. Parkin exists in an auto-inhibited conformation stabilized by multiple interdomain interactions. The binding of PINK1-generated phospho-ubiquitin and the phosphorylation of the ubiquitin-like (Ubl) domain of Parkin at Ser65 release its auto-inhibition, but how and when these events take place in cells remain to be defined. Here we show that mutations that we designed to activate Parkin by releasing the Repressor Element of Parkin (REP) domain, or by disrupting the interface between the RING0:RING2 domains, can completely rescue mutations in the Parkin Ubl that are defective in mitochondrial autophagy. Using a FRET reporter assay we show that Parkin undergoes a conformational change upon phosphorylation that can be mimicked by mutating Trp403 in the REP. We propose a hierarchical model whereby pUb binding on mitochondria enables Parkin phosphorylation, which, in turn, leads to REP removal, E3 ligase activation and mitophagy. PMID:28276439

Structure-guided mutagenesis reveals a hierarchical mechanism of Parkin activation.

PubMed

Tang, Matthew Y; Vranas, Marta; Krahn, Andrea I; Pundlik, Shayal; Trempe, Jean-François; Fon, Edward A

2017-03-09

Parkin and PINK1 function in a common pathway to clear damaged mitochondria. Parkin exists in an auto-inhibited conformation stabilized by multiple interdomain interactions. The binding of PINK1-generated phospho-ubiquitin and the phosphorylation of the ubiquitin-like (Ubl) domain of Parkin at Ser65 release its auto-inhibition, but how and when these events take place in cells remain to be defined. Here we show that mutations that we designed to activate Parkin by releasing the Repressor Element of Parkin (REP) domain, or by disrupting the interface between the RING0:RING2 domains, can completely rescue mutations in the Parkin Ubl that are defective in mitochondrial autophagy. Using a FRET reporter assay we show that Parkin undergoes a conformational change upon phosphorylation that can be mimicked by mutating Trp403 in the REP. We propose a hierarchical model whereby pUb binding on mitochondria enables Parkin phosphorylation, which, in turn, leads to REP removal, E3 ligase activation and mitophagy.

Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions

PubMed Central

2015-01-01

Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein–protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein–protein interactions within whole animals. PMID:25265085

Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

PubMed

Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

2015-05-15

Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

Structural basis for collagen recognition by the immune receptor OSCAR.

PubMed

Zhou, Long; Hinerman, Jennifer M; Blaszczyk, Michal; Miller, Jeanette L C; Conrady, Deborah G; Barrow, Alexander D; Chirgadze, Dimitri Y; Bihan, Dominique; Farndale, Richard W; Herr, Andrew B

2016-02-04

The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2. © 2016 by The American Society of Hematology.

New insights into apoptosome structure and function.

PubMed

Dorstyn, Loretta; Akey, Christopher W; Kumar, Sharad

2018-05-15

The apoptosome is a platform that activates apical procaspases in response to intrinsic cell death signals. Biochemical and structural studies in the past two decades have extended our understanding of apoptosome composition and structure, while illuminating the requirements for initiator procaspase activation. A number of studies have now provided high-resolution structures for apoptosomes from C. elegans (CED-4), D. melanogaster (Dark), and H. sapiens (Apaf-1), which define critical protein interfaces, including intra and interdomain interactions. This work also reveals interactions of apoptosomes with their respective initiator caspases, CED-3, Dronc and procaspase-9. Structures of the human apoptosome have defined the requirements for cytochrome c binding, which triggers the conversion of inactive Apaf-1 molecules to an extended, assembly competent state. While recent data have provided a detailed understanding of apoptosome formation and procaspase activation, they also highlight important evolutionary differences with functional implications for caspase activation. CARD/CARD interactions in the CED-4, Dark and Apaf-1 apoptosomes. Type I, II and III interfaces that stabilize CARD-CARD interactions are indicated (left column). Note that the Type I interface appears to be unique to Apaf-1/pc-9 CARD interactions. Middle column shows cartoons of the active states of the CARD-CARD disks, illustrating the two CED-4 tetrameric ring layers (top) and the recruitment of 8 Dronc CARDs and between 3-4 pc-9 CARDs, to the Dark and Apaf-1 apoptosomes respectively (middle and lower panels). Ribbon diagrams of the CED-4, Dark and Apaf-1 apoptosomes are shown (right column).

Solving Kinetic Equations for the Laser Flash Photolysis Experiment on Nitric Oxide Synthases: Effect of Conformational Dynamics on the Interdomain Electron Transfer.

PubMed

Astashkin, Andrei V; Feng, Changjian

2015-11-12

The production of nitric oxide by the nitric oxide synthase (NOS) enzyme depends on the interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme domains. Although the rate of this IET has been measured by laser flash photolysis (LFP) for various NOS proteins, no rigorous analysis of the relevant kinetic equations was performed so far. In this work, we provide an analytical solution of the kinetic equations underlying the LFP approach. The derived expressions reveal that the bulk IET rate is significantly affected by the conformational dynamics that determines the formation and dissociation rates of the docking complex between the FMN and heme domains. We show that in order to informatively study the electron transfer across the NOS enzyme, LFP should be used in combination with other spectroscopic methods that could directly probe the docking equilibrium and the conformational change rate constants. The implications of the obtained analytical expressions for the interpretation of the LFP results from various native and modified NOS proteins are discussed. The mathematical formulas derived in this work should also be applicable for interpreting the IET kinetics in other modular redox enzymes.

Size, Shape, and Lateral Correlation of Highly Uniform, Mesoscopic, Self-Assembled Domains of Fluorocarbon-Hydrocarbon Diblocks at the Air/Water Interface: A GISAXS Study.

PubMed

Veschgini, Mariam; Abuillan, Wasim; Inoue, Shigeto; Yamamoto, Akihisa; Mielke, Salomé; Liu, Xianhe; Konovalov, Oleg; Krafft, Marie Pierre; Tanaka, Motomu

2017-10-06

The shape and size of self-assembled mesoscopic surface domains of fluorocarbon-hydrocarbon (FnHm) diblocks and the lateral correlation between these domains were quantitatively determined from grazing incidence small-angle X-ray scattering (GISAXS). The full calculation of structure and form factors unravels the influence of fluorocarbon and hydrocarbon block lengths on the diameter and height of the domains, and provides the inter-domain correlation length. The diameter of the domains, as determined from the form factor analysis, exhibits a monotonic increase in response to the systematic lengthening of each block, which can be attributed to the increase in van der Waals attraction between molecules. The pair correlation function in real space calculated from the structure factor implies that the inter-domain correlation can reach a distance that is over 25 times larger than the domain's size. The full calculation of the GISAXS signals introduced here opens a potential towards the hierarchical design of mesoscale domains of self-assembled small organic molecules, covering several orders of magnitude in space. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional characteristics of the calcium modulated proteins seen from an evolutionary perspective

NASA Technical Reports Server (NTRS)

Kretsinger, R. H.; Nakayama, S.; Moncrief, N. D.

1991-01-01

We have constructed dendrograms relating 173 EF-hand proteins of known amino acid sequence. We aligned all of these proteins by their EF-hand domains, omitting interdomain regions. Initial dendrograms were computed by minimum mutation distance methods. Using these as starting points, we determined the best dendrogram by the method of maximum parsimony, scored by minimum mutation distance. We identified 14 distinct subfamilies as well as 6 unique proteins that are perhaps the sole representatives of other subfamilies. This information is given in tabular form. Within subfamilies one can easily align interdomain regions. The resulting dendrograms are very similar to those computed using domains only. Dendrograms constructed using pairs of domains show general congruence. However, there are enough exceptions to caution against an overly simple scheme in which one pair of gene duplications leads from one domain precurser to a four domain prototype from which all other forms evolved. The ability to bind calcium was lost and acquired several times during evolution. The distribution of introns does not conform to the dendrogram based on amino acid sequences. The rates of evolution appear to be much slower within subfamilies, especially within calmodulin, than those prior to the definition of subfamily.

Induced-fit Mechanism for Prolyl Endopeptidase

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

Li, Min; Chen, Changqing; Davies, David R.

2010-11-15

Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the {beta}-propeller and {alpha}/{beta}-hydrolase domains; addition of substrate tomore » preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.« less

Slow Domain Motions of an Oligomeric Protein from Deep-Sea Hyperthermophile probed by Neutron Spin Echo

NASA Astrophysics Data System (ADS)

Bhowmik, Debsindhu; Shrestha, Utsab; Dhindsa, Gurpreet; Sharp, Melissa; Stingaciu, Laura R.; Chu, Xiang-Qiang; Xiang-Qiang Chu Team

Deep-sea microorganisms have the ability to survive under extreme conditions, such as high pressure and high temperature. In this work, we used the combination of the neutron spin-echo (NSE) and the small angle neutron scattering (SANS) techniques to study the inter-domain motions of the inorganic pyrophosphate (IPPase) enzyme derived from thermostable microorganisms Thermococcus thioreducens. The IPPase has hexameric quaternary structure with molecular mass of approx. 120kDa (each subunit of 20kDa), which is a large oligomeric structure. The understanding of its slow inter-domain motions can be the key to explain how they are able to perform catalytic activity at higher temperature compared to mesophilic enzymes, thus leading to adapt to extreme environment present at the seabed. The NSE can probe these slow motions directly in the time domain up to several tens of nanoseconds at the nanometers length scales, while the corresponding structural change can be explored by the SANS. Our results provide a better picture of the local flexibility and conformational substates unique to these types of proteins, which will help us better understandthe relation between protein dynamics and their biological activities

Probing Allosteric Inhibition Mechanisms of the Hsp70 Chaperone Proteins Using Molecular Dynamics Simulations and Analysis of the Residue Interaction Networks.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2016-08-22

Although molecular mechanisms of allosteric regulation in the Hsp70 chaperones have been extensively studied at both structural and functional levels, the current understanding of allosteric inhibition of chaperone activities by small molecules is still lacking. In the current study, using a battery of computational approaches, we probed allosteric inhibition mechanisms of E. coli Hsp70 (DnaK) and human Hsp70 proteins by small molecule inhibitors PET-16 and novolactone. Molecular dynamics simulations and binding free energy analysis were combined with network-based modeling of residue interactions and allosteric communications to systematically characterize and compare molecular signatures of the apo form, substrate-bound, and inhibitor-bound chaperone complexes. The results suggested a mechanism by which the allosteric inhibitors may leverage binding energy hotspots in the interaction networks to stabilize a specific conformational state and impair the interdomain allosteric control. Using the network-based centrality analysis and community detection, we demonstrated that substrate binding may strengthen the connectivity of local interaction communities, leading to a dense interaction network that can promote an efficient allosteric communication. In contrast, binding of PET-16 to DnaK may induce significant dynamic changes and lead to a fractured interaction network and impaired allosteric communications in the DnaK complex. By using a mechanistic-based analysis of distance fluctuation maps and allosteric propensities of protein residues, we determined that the allosteric network in the PET-16 complex may be small and localized due to the reduced communication and low cooperativity of the substrate binding loops, which may promote the higher rates of substrate dissociation and the decreased substrate affinity. In comparison with the significant effect of PET-16, binding of novolactone to HSPA1A may cause only moderate network changes and preserve allosteric coupling between the allosteric pocket and the substrate binding region. The impact of novolactone on the conformational dynamics and allosteric communications in the HSPA1A complex was comparable to the substrate effect, which is consistent with the experimental evidence that PET-16, but not novolactone binding, can significantly decrease substrate affinity. We argue that the unique dynamic and network signatures of PET-16 and novolactone may be linked with the experimentally observed functional effects of these inhibitors on allosteric regulation and substrate binding.

Global Organization of a Positive-strand RNA Virus Genome

PubMed Central

Wu, Baodong; Grigull, Jörg; Ore, Moriam O.; Morin, Sylvie; White, K. Andrew

2013-01-01

The genomes of plus-strand RNA viruses contain many regulatory sequences and structures that direct different viral processes. The traditional view of these RNA elements are as local structures present in non-coding regions. However, this view is changing due to the discovery of regulatory elements in coding regions and functional long-range intra-genomic base pairing interactions. The ∼4.8 kb long RNA genome of the tombusvirus tomato bushy stunt virus (TBSV) contains these types of structural features, including six different functional long-distance interactions. We hypothesized that to achieve these multiple interactions this viral genome must utilize a large-scale organizational strategy and, accordingly, we sought to assess the global conformation of the entire TBSV genome. Atomic force micrographs of the genome indicated a mostly condensed structure composed of interconnected protrusions extending from a central hub. This configuration was consistent with the genomic secondary structure model generated using high-throughput selective 2′-hydroxyl acylation analysed by primer extension (i.e. SHAPE), which predicted different sized RNA domains originating from a central region. Known RNA elements were identified in both domain and inter-domain regions, and novel structural features were predicted and functionally confirmed. Interestingly, only two of the six long-range interactions known to form were present in the structural model. However, for those interactions that did not form, complementary partner sequences were positioned relatively close to each other in the structure, suggesting that the secondary structure level of viral genome structure could provide a basic scaffold for the formation of different long-range interactions. The higher-order structural model for the TBSV RNA genome provides a snapshot of the complex framework that allows multiple functional components to operate in concert within a confined context. PMID:23717202

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense.

PubMed

Nishikawa, C Y; Araújo, L M; Kadowaki, M A S; Monteiro, R A; Steffens, M B R; Pedrosa, F O; Souza, E M; Chubatsu, L S

2012-02-01

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ(54) co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH(4)Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

PubMed Central

Nishikawa, C.Y.; Araújo, L.M.; Kadowaki, M.A.S.; Monteiro, R.A.; Steffens, M.B.R.; Pedrosa, F.O.; Souza, E.M.; Chubatsu, L.S.

2012-01-01

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription. PMID:22267004

Ligand-induced perturbation of the HIF-2α:ARNT dimer dynamics

PubMed Central

Motta, Stefano

2018-01-01

Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix−loop−helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses. PMID:29489822

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

Lolicato, Marco; Arrigoni, Cristina; Mori, Takahiro

Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) channels of the TREK1 subfamily generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses1, 2, 3. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit4, 5, 6. In contrast to other potassium channels, K2P channels use a selectivity filter ‘C-type’ gate7, 8, 9, 10 as the principal gating site. Despite recent advances3, 11, 12, poor pharmacological profiles of K2P channels limit mechanistic and biological studies. Here we describe a classmore » of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators—an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402—define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation–π interaction that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter gating.« less

Explaining the Substantial Inter-Domain and Over-Time Correlations in Student Achievement: The Importance of Stable Student Attributes

ERIC Educational Resources Information Center

Marks, Gary N.

2016-01-01

Multi-domain and longitudinal studies of student achievement routinely find moderate to strong correlations across achievement domains and even stronger within-domain correlations over time. The purpose of this study is to examine the sources of these patterns analysing student achievement in 5 domains across Years 3, 5 and 7. The analysis is of…

A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

DOE PAGES

Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; ...

2016-05-03

Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain amore » canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

The Molecular Dynamics Study of the Structural Conversions in the Transformer Protein RfaH

NASA Astrophysics Data System (ADS)

Gc, Jeevan; Gerstman, Bernard; Chapagain, Prem

Recently, a class of multi-domain proteins such as RfaH transcription factor are labelled as the transformer proteins as they undergo major conformational transformation for performing multiple functions. In the absence of the inter-domain contacts, the C-terminal domain of RfaH transforms from its alpha-helix conformation to a beta-barrel structure. Each of these states have their own functional role: in its alpha-helx state, RfaH-CTD inhibits the transcription by masking the binding site of RNAP, but in its beta state it facilitates the translation. We used various molecular dynamics simulations to study its transformer-like behavior of full-RfaH and identified key amino acid residues that are important in modulating such behavior. Our results show that the inter domain interactions constitute the major barrier in the alpha-helix to beta-barrel conversion. Once the interfacial interactions are broken, structural conversion is easier. The structural conversion from beta-barrel to alpha-helix proceeds with the rearrangement of the hydrophobic residues followed by the inter domain contacts formation via non-native, transient salt-bridge formation, leading to the formation of the native inter domain salt-bridge and hydrophobic contacts to give the final alpha-helix structure.

Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves.

PubMed

Baladi, S; Tsvetkov, P O; Petrova, T V; Takagi, T; Sakamoto, H; Lobachov, V M; Makarov, A A; Cox, J A

2001-04-01

Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N-CaVP) and carboxy-terminal halves (C-CaVP). The interactive properties of recombinant and wild-type CaVP are very similar, despite three post-translational modifications in the wild-type protein. N-CaVP does not bind Ca2+, shows a well-formed hydrophobic core, and melts at 44 degrees C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 microM (i.e., very similar to the entire CaVP). The metal-free domain in CaVP and C-CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+) forms melt in the 111 degrees -123 degrees C range, suggesting that C-CaVP and the carboxy- domain of CaVP are natively unfolded in the metal-free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy-domain destabilize to different degrees the amino-domain. Only C-CaVP forms a Ca2+-dependent 1:1 complex with CaVPT. Our results suggest that the carboxy-terminal domain of CaVP interacts with CaVPT and that the amino-terminal lobe modulates this interaction.

Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves

PubMed Central

Baladi, Sibyl; Tsvetkov, Philipp O.; Petrova, Tatiana V.; Takagi, Takashi; Sakamoto, Hiroshi; Lobachov, Vladimir M.; Makarov, Alexander A.; Cox, Jos A.

2001-01-01

Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N-CaVP) and carboxy-terminal halves (C-CaVP). The interactive properties of recombinant and wild-type CaVP are very similar, despite three post-translational modifications in the wild-type protein. N-CaVP does not bind Ca2+, shows a well-formed hydrophobic core, and melts at 44°C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 μM (i.e., very similar to the entire CaVP). The metal-free domain in CaVP and C-CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+ forms melt in the 111°–123°C range, suggesting that C-CaVP and the carboxy- domain of CaVP are natively unfolded in the metal-free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy-domain destabilize to different degrees the amino-domain. Only C-CaVP forms a Ca2+-dependent 1:1 complex with CaVPT. Our results suggest that the carboxy-terminal domain of CaVP interacts with CaVPT and that the amino-terminal lobe modulates this interaction. PMID:11274468

Exploration of Structural Changes in Lactose Permease on Sugar Binding and Proton Transport through Atomistic Simulations

NASA Astrophysics Data System (ADS)

Liu, Jin; Jewel, Yead; Dutta, Prashanta

2017-11-01

Escherichia coli lactose permease (LacY) actively transports lactose and other galactosides across cell membranes through lactose/H+ symport process. Lactose/H+ symport is a highly complex process that involves large-scale protein conformational changes. The complete picture of lactose/H+ symport is largely unclear. In this work, we develop the force field for sugar molecules compatible with PACE, a hybrid and coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid. After validation, we implement the new force field to investigate the binding of a β-D-galactopyranosyl-1-thio- β-D-galactopyranoside (TDG) molecule to a wild-type LacY. Transitions from inward-facing to outward-facing conformations upon TDG binding and protonation of Glu269 have been achieved from microsecond simulations. Both the opening of the periplasmic side and closure of the cytoplasmic side of LacY are consistent with experiments. Our analysis suggest that the conformational changes of LacY are a cumulative consequence of inter-domain H-bonds breaking at the periplasmic side, inter-domain salt-bridge formation at the cytoplasmic side, as well as the TDG orientational changes during the transition. This work is supported by US National Science Foundation under Grant No. CBET-1604211.

Scaling the PuNDIT project for wide area deployments

NASA Astrophysics Data System (ADS)

McKee, Shawn; Batista, Jorge; Carcassi, Gabriele; Dovrolis, Constantine; Lee, Danny

2017-10-01

In today’s world of distributed scientific collaborations, there are many challenges to providing reliable inter-domain network infrastructure. Network operators use a combination of active monitoring and trouble tickets to detect problems, but these are often ineffective at identifying issues that impact wide-area network users. Additionally, these approaches do not scale to wide area inter-domain networks due to unavailability of data from all the domains along typical network paths. The Pythia Network Diagnostic InfrasTructure (PuNDIT) project aims to create a scalable infrastructure for automating the detection and localization of problems across these networks. The project goal is to gather and analyze metrics from existing perfSONAR monitoring infrastructures to identify the signatures of possible problems, locate affected network links, and report them to the user in an intuitive fashion. Simply put, PuNDIT seeks to convert complex network metrics into easily understood diagnoses in an automated manner. We present our progress in creating the PuNDIT system and our status in developing, testing and deploying PuNDIT. We report on the project progress to-date, describe the current implementation architecture and demonstrate some of the various user interfaces it will support. We close by discussing the remaining challenges and next steps and where we see the project going in the future.

Toughening of a Carbon-Fibre Composite Using Electrospun Poly(Hydroxyether of Bisphenol A) Nanofibrous Membranes Through Inverse Phase Separation and Inter-Domain Etherification

PubMed Central

Magniez, Kevin; Chaffraix, Thomas; Fox, Bronwyn

2011-01-01

The interlaminar toughening of a carbon fibre reinforced composite by interleaving a thin layer (~20 microns) of poly(hydroxyether of bisphenol A) (phenoxy) nanofibres was explored in this work. Nanofibres, free of defect and averaging several hundred nanometres, were produced by electrospinning directly onto a pre-impregnated carbon fibre material (Toray G83C) at various concentrations between 0.5 wt % and 2 wt %. During curing at 150 °C, phenoxy diffuses through the epoxy resin to form a semi interpenetrating network with an inverse phase type of morphology where the epoxy became the co-continuous phase with a nodular morphology. This type of morphology improved the fracture toughness in mode I (opening failure) and mode II (in-plane shear failure) by up to 150% and 30%, respectively. Interlaminar shear stress test results showed that the interleaving did not negatively affect the effective in-plane strength of the composites. Furthermore, there was some evidence from DMTA and FT-IR analysis to suggest that inter-domain etherification between the residual epoxide groups with the pendant hydroxyl groups of the phenoxy occurred, also leading to an increase in glass transition temperature (~7.5 °C). PMID:28824118

The mediator complex in genomic and non-genomic signaling in cancer.

PubMed

Weber, Hannah; Garabedian, Michael J

2018-05-01

Mediator is a conserved, multi-subunit macromolecular machine divided structurally into head, middle, and tail modules, along with a transiently associating kinase module. Mediator functions as an integrator of transcriptional regulatory activity by interacting with DNA-bound transcription factors and with RNA polymerase II (Pol II) to both activate and repress gene expression. Mediator has been shown to affect multiple steps in transcription, including chromatin looping between enhancers and promoters, pre-initiation complex formation, transcriptional elongation, and mRNA splicing. Individual Mediator subunits participate in regulation of gene expression by the estrogen and androgen receptors and are altered in a number of endocrine cancers, including breast and prostate cancer. In addition to its role in genomic signaling, MED12 has been implicated in non-genomic signaling by interacting with and activating TGF-beta receptor 2 in the cytoplasm. Recent structural studies have revealed extensive inter-domain interactions and complex architecture of the Mediator-Pol II complex, suggesting that Mediator is capable of reorganizing its conformation and composition to fit cellular needs. We propose that alterations in Mediator subunit expression that occur in various cancers could impact the organization and function of Mediator, resulting in changes in gene expression that promote malignancy. A better understanding of the role of Mediator in cancer could reveal new approaches to the diagnosis and treatment of Mediator-dependent endocrine cancers, especially in settings of therapy resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Rapid fabrication of hierarchically structured supramolecular nanocomposite thin films in one minute

DOEpatents

Xu, Ting; Kao, Joseph

2016-11-08

Functional nanocomposites containing nanoparticles of different chemical compositions may exhibit new properties to meet demands for advanced technology. It is imperative to simultaneously achieve hierarchical structural control and to develop rapid, scalable fabrication to minimize degradation of nanoparticle properties and for compatibility with nanomanufacturing. The assembly kinetics of supramolecular nanocomposite in thin films is governed by the energetic cost arising from defects, the chain mobility, and the activation energy for inter-domain diffusion. By optimizing only one parameter, the solvent fraction in the film, the assembly kinetics can be precisely tailored to produce hierarchically structured thin films of supramolecular nanocomposites in approximately one minute. Moreover, the strong wavelength dependent optical anisotropy in the nanocomposite highlights their potential applications for light manipulation and information transmission. The present invention opens a new avenue in designing manufacture-friendly continuous processing for the fabrication of functional nanocomposite thin films.

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

Hood, Iris V.; Berger, James M.

Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases. How loader activity is appropriately controlled remains unclear. Here, we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of theStaphylococcus aureusreplicative helicase loader, DnaI. The viral protein binds to the loader’s AAA+ ATPase domain, allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity. Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins. We find that the inhibitor protein is genetically coupled tomore » a phage-encoded homolog of the bacterial helicase loader, which we show binds to the host helicase but not to the inhibitor itself. These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association.« less

Health Information System Role-Based Access Control Current Security Trends and Challenges.

PubMed

de Carvalho Junior, Marcelo Antonio; Bandiera-Paiva, Paulo

2018-01-01

This article objective is to highlight implementation characteristics, concerns, or limitations over role-based access control (RBAC) use on health information system (HIS) using industry-focused literature review of current publishing for that purpose. Based on the findings, assessment for indication of RBAC is obsolete considering HIS authorization control needs. We have selected articles related to our investigation theme "RBAC trends and limitations" in 4 different sources related to health informatics or to the engineering technical field. To do so, we have applied the following search query string: "Role-Based Access Control" OR "RBAC" AND "Health information System" OR "EHR" AND "Trends" OR "Challenges" OR "Security" OR "Authorization" OR "Attacks" OR "Permission Assignment" OR "Permission Relation" OR "Permission Mapping" OR "Constraint". We followed PRISMA applicable flow and general methodology used on software engineering for systematic review. 20 articles were selected after applying inclusion and exclusion criteria resulting contributions from 10 different countries. 17 articles advocate RBAC adaptations. The main security trends and limitations mapped were related to emergency access, grant delegation, and interdomain access control. Several publishing proposed RBAC adaptations and enhancements in order to cope current HIS use characteristics. Most of the existent RBAC studies are not related to health informatics industry though. There is no clear indication of RBAC obsolescence for HIS use.

Crystal structure of arrestin-3 reveals the basis of the difference in receptor binding between two non-visual subtypes

PubMed Central

Zhan, Xuanzhi; Gimenez, Luis E.; Gurevich, Vsevolod V.; Spiller, Benjamin W.

2011-01-01

Arrestins are multi-functional proteins that regulate signaling and trafficking of the majority of G protein-coupled receptors (GPCRs), as well as sub-cellular localization and activity of many other signaling proteins. Here we report the first crystal structure of arrestin-3, solved at 3.0Å. Arrestin-3 is an elongated two-domain molecule with the overall fold and key inter-domain interactions that hold free protein in the basal conformation similar to the other subtypes. Arrestin-3 is the least selective member of the family, binding wide variety of GPCRs with high affinity and demonstrating lower preference for active phosphorylated forms of the receptors. In contrast to the other three arrestins, part of the receptor-binding surface in the arrestin-3 C-domain does not form a contiguous β-sheet, consistent with increased flexibility. By swapping the corresponding elements between arrestin-2 and -3 we show that the presence of this loose structure correlates with reduced arrestin selectivity for activated receptor, consistent with a conformational change in this β-sheet upon receptor binding. PMID:21215759

Structural features of Fab fragments of rheumatoid factor IgM-RF in solution

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

Volkov, V. V., E-mail: vvo@ns.crys.ras.ru; Lapuk, V. A.; Shtykova, E. V.

The structural features of the Fab fragments of monoclonal (Waldenstroem's disease) immunoglobulin M (IgM) and rheumatoid immunoglobulin M (IgM-RF) were studied by a complex of methods, including small-angle X-ray scattering (SAXS), electron spin resonance (ESR), and mass spectrometry (MS). The Fab-RF fragment was demonstrated to be much more flexible in the region of interdomain contacts, the molecular weights and the shapes of the Fab and Fab-RF macromolecules in solution being only slightly different. According to the ESR data, the rotational correlation time for a spin label introduced into the peptide sequence for Fab is twice as large as that formore » Fab-RF (21{+-}2 and 11{+-}1 ns, respectively), whereas the molecular weights of these fragments differ by only 0.5% (mass-spectrometric data), which correlates with the results of molecular-shape modeling by small-angle X-ray scattering. The conclusion about the higher flexibility of the Fab-RF fragment contributes to an understanding of the specificity of interactions between the rheumatoid factor and the antigens of the own organism.« less

Evolution of thermophilic DNA polymerases for the recognition and amplification of C2ʹ-modified DNA

NASA Astrophysics Data System (ADS)

Chen, Tingjian; Hongdilokkul, Narupat; Liu, Zhixia; Adhikary, Ramkrishna; Tsuen, Shujian S.; Romesberg, Floyd E.

2016-06-01

The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, in many applications the use of these aptamers is limited by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2ʹ positions with methoxy or fluorine substituents are stable to nucleases, but they cannot be synthesized by natural polymerases. Here we report the development of a polymerase-evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2ʹ-OMe-modified oligonucleotides and their DNA counterparts via ‘transcription’ and ‘reverse transcription’ or, more importantly, that PCR-amplify partially C2ʹ-OMe- or C2ʹ-F-modified oligonucleotides. A mechanistic analysis demonstrates that the ability to amplify the modified oligonucleotides evolved by optimizing interdomain interactions that stabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practical applications and the developed evolution system should be a powerful tool for tailoring polymerases to have other types of novel function.

Structural and Biochemical Insights into the Multiple Functions of Yeast Grx3.

PubMed

Chi, Chang-Biao; Tang, YaJun; Zhang, Jiahai; Dai, Ya-Nan; Abdalla, Mohnad; Chen, Yuxing; Zhou, Cong-Zhao

2018-04-13

The yeast Saccharomyces cerevisiae monothiol glutaredoxin Grx3 plays a key role in cellular defense against oxidative stress and more importantly, cooperates with BolA-like iron repressor of activation protein Fra2 to regulate the localization of the iron-sensing transcription factor Aft2. The interplay among Grx3, Fra2 and Aft2 responsible for the regulation of iron homeostasis has not been clearly described. Here we solved the crystal structures of the Trx domain (Grx3 Trx ) and Grx domain (Grx3 Grx ) of Grx3 in addition to the solution structure of Fra2. Structural analyses and activity assays indicated that the Trx domain also contributes to the glutathione S-transferase activity of Grx3, via an inter-domain disulfide bond between Cys37 and Cys176. NMR titration and pull-down assays combined with surface plasmon resonance experiments revealed that Fra2 could form a noncovalent heterodimer with Grx3 via an interface between the helix-turn-helix motif of Fra2 and the C-terminal segment of Grx3 Grx , different from the previously identified covalent heterodimer mediated by Fe-S cluster. Comparative affinity assays indicated that the interaction between Fra2 and Aft2 is much stronger than that between Grx3 and Aft2, or Aft2 toward its target DNA. These structural and biochemical analyses enabled us to propose a model how Grx3 executes multiple functions to coordinate the regulation of Aft2-controlled iron metabolism. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mechanism of allosteric inhibition of N-acetyl-L-glutamate synthase by L-arginine.

PubMed

Min, Li; Jin, Zhongmin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

2009-02-20

N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in l-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by l-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with l-arginine bound and in the active R-state complexed with CoA and l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of l-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by approximately 10 A and decreases its height by approximately 20A(.) AAK dimers move 5A outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by approximately 4 degrees . The NAT domains rotate approximately 109 degrees relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the l-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

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

Min, Li; Jin, Zhongmin; Caldovic, Ljubica

2010-01-07

N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in L-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by L-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with L-arginine bound and in the active R-state complexed with CoA and L-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of L-arginine to the AAKmore » domain induces a global conformational change that increases the diameter of the hexamer by {approx}10 {angstrom} and decreases its height by {approx}20{angstrom}. AAK dimers move 5{angstrom} outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by {approx}4{sup o}. The NAT domains rotate {approx}109{sup o} relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the L-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.« less

High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p

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

Mohr, Georg; Del Campo, Mark; Turner, Kathryn G.

The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionallymore » important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions - one the previously noted post II region in the helicase core and the other in the CTE - that may help displace or sequester the opposite RNA strand during RNA unwinding.« less

A Failure to Communicate

PubMed Central

Kronert, William A.; Melkani, Girish C.; Melkani, Anju; Bernstein, Sanford I.

2015-01-01

Our molecular modeling studies suggest a charge-dependent interaction between residues Glu-497 in the relay domain and Arg-712 in the converter domain of human β-cardiac myosin. To test the significance of this putative interaction, we generated transgenic Drosophila expressing indirect flight muscle myosin with charge reversal mutations in the relay (E496R) or converter (R713E). Each mutation yielded dramatic reductions in myosin Ca-ATPase activity (∼80%) as well as in basal (∼67%) and actin-activated (∼84%) Mg-ATPase activity. E496R myosin-induced in vitro actin-sliding velocity was reduced by 71% and R713E myosin permitted no actin motility. Indirect flight muscles of late pupae from each mutant displayed disrupted myofibril assembly, with adults having severely abnormal myofibrils and no flight ability. To understand the molecular basis of these defects, we constructed a putative compensatory mutant that expresses myosin with both E496R and R713E. Intriguingly, ATPase values were restored to ∼73% of wild-type and actin-sliding velocity increased to 40%. The double mutation suppresses myofibril assembly defects in pupal indirect flight muscles and dramatically reduces myofibril disruption in young adults. Although sarcomere organization is not sustained in older flies and flight ability is not restored in homozygotes, young heterozygotes fly well. Our results indicate that this charge-dependent interaction between the myosin relay and converter domains is essential to the mechanochemical cycle and sarcomere assembly. Furthermore, the same inter-domain interaction is disrupted when modeling human β-cardiac myosin heavy chain cardiomyopathy mutations E497D or R712L, implying that abolishing this salt bridge is one cause of the human disease. PMID:26446785

Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?

PubMed Central

Gil-Moreno, Selene; Jiménez-Martí, Elena; Palacios, Òscar; Zerbe, Oliver; Dallinger, Reinhard; Capdevila, Mercè; Atrian, Sílvia

2015-01-01

Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metal ions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion. PMID:26703589

A structured interdomain linker directs self-polymerization of human uromodulin

PubMed Central

Bokhove, Marcel; Nishimura, Kaoru; Brunati, Martina; Han, Ling; de Sanctis, Daniele; Rampoldi, Luca

2016-01-01

Uromodulin (UMOD)/Tamm–Horsfall protein, the most abundant human urinary protein, plays a key role in chronic kidney diseases and is a promising therapeutic target for hypertension. Via its bipartite zona pellucida module (ZP-N/ZP-C), UMOD forms extracellular filaments that regulate kidney electrolyte balance and innate immunity, as well as protect against renal stones. Moreover, salt-dependent aggregation of UMOD filaments in the urine generates a soluble molecular net that captures uropathogenic bacteria and facilitates their clearance. Despite the functional importance of its homopolymers, no structural information is available on UMOD and how it self-assembles into filaments. Here, we report the crystal structures of polymerization regions of human UMOD and mouse ZP2, an essential sperm receptor protein that is structurally related to UMOD but forms heteropolymers. The structure of UMOD reveals that an extensive hydrophobic interface mediates ZP-N domain homodimerization. This arrangement is required for filament formation and is directed by an ordered ZP-N/ZP-C linker that is not observed in ZP2 but is conserved in the sequence of deafness/Crohn’s disease-associated homopolymeric glycoproteins α-tectorin (TECTA) and glycoprotein 2 (GP2). Our data provide an example of how interdomain linker plasticity can modulate the function of structurally similar multidomain proteins. Moreover, the architecture of UMOD rationalizes numerous pathogenic mutations in both UMOD and TECTA genes. PMID:26811476

Models for randomly distributed nanoscopic domains on spherical vesicles

NASA Astrophysics Data System (ADS)

Anghel, Vinicius N. P.; Bolmatov, Dima; Katsaras, John

2018-06-01

The existence of lipid domains in the plasma membrane of biological systems has proven controversial, primarily due to their nanoscopic size—a length scale difficult to interrogate with most commonly used experimental techniques. Scattering techniques have recently proven capable of studying nanoscopic lipid domains populating spherical vesicles. However, the development of analytical methods able of predicting and analyzing domain pair correlations from such experiments has not kept pace. Here, we developed models for the random distribution of monodisperse, circular nanoscopic domains averaged on the surface of a spherical vesicle. Specifically, the models take into account (i) intradomain correlations corresponding to form factors and interdomain correlations corresponding to pair distribution functions, and (ii) the analytical computation of interdomain correlations for cases of two and three domains on a spherical vesicle. In the case of more than three domains, these correlations are treated either by Monte Carlo simulations or by spherical analogs of the Ornstein-Zernike and Percus-Yevick (PY) equations. Importantly, the spherical analog of the PY equation works best in the case of nanoscopic size domains, a length scale that is mostly inaccessible by experimental approaches such as, for example, fluorescent techniques and optical microscopies. The analytical form factors and structure factors of nanoscopic domains populating a spherical vesicle provide a new and important framework for the quantitative analysis of experimental data from commonly studied phase-separated vesicles used in a wide range of biophysical studies.

Reconstructed Ancestral Myo-Inositol-3-Phosphate Synthases Indicate That Ancestors of the Thermococcales and Thermotoga Species Were More Thermophilic than Their Descendants

PubMed Central

Butzin, Nicholas C.; Lapierre, Pascal; Green, Anna G.; Swithers, Kristen S.; Gogarten, J. Peter; Noll, Kenneth M.

2013-01-01

The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS). These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT) of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants. PMID:24391933

Computational Modeling of Allosteric Regulation in the Hsp90 Chaperones: A Statistical Ensemble Analysis of Protein Structure Networks and Allosteric Communications

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2014-01-01

A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks. PMID:24922508

Computational modeling of allosteric regulation in the hsp90 chaperones: a statistical ensemble analysis of protein structure networks and allosteric communications.

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-06-01

A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks.

Multinational Experiment 7. Outcome 4: Understanding Inter-Domain Dependencies and Vulnerabilities. Conceptual and Pre-Doctrinal paper

DTIC Science & Technology

2013-01-31

military force projection. The ACT “Assured Access to the Global Commons” report for example states that “in the coming decade the Alliance will face an...strength and success comes from its ability to use the Global Commons in accordance with international law”9. Yet, new complexities in the Global...the future it may also be provided by Galileo , GLONASS or BEIDOU. 22 In the future it may also be provided by Galileo , GLONASS or BEIDOU. UNCLASSIFIED

A Linked-Cell Domain Decomposition Method for Molecular Dynamics Simulation on a Scalable Multiprocessor

DOE PAGES

Yang, L. H.; Brooks III, E. D.; Belak, J.

1992-01-01

A molecular dynamics algorithm for performing large-scale simulations using the Parallel C Preprocessor (PCP) programming paradigm on the BBN TC2000, a massively parallel computer, is discussed. The algorithm uses a linked-cell data structure to obtain the near neighbors of each atom as time evoles. Each processor is assigned to a geometric domain containing many subcells and the storage for that domain is private to the processor. Within this scheme, the interdomain (i.e., interprocessor) communication is minimized.

Bulk YBa2Cu3O(x) superconductors through pressurized partial melt growth processing

NASA Technical Reports Server (NTRS)

Hu, S.; Hojaji, H.; Barkatt, A.; Boroomand, M.; Hung, M.; Buechele, A. C.; Thorpe, A. N.; Davis, D. D.; Alterescu, S.

1992-01-01

A novel pressurized partial melt growth process has been developed for producing large pieces of bulk Y-Ba-Cu-O superconductors. During long-time partial melt growth stage, an additional driving force for solidification is obtained by using pressurized oxygen gas. The microstructure and superconducting properties of the resulting samples were investigated. It was found that this new technique can eliminate porosity and inhomogeneity, promote large-scale grain-texturing, and improve interdomain coupling as well.

Electro-chemical coupling in the voltage-dependent phosphatase Ci-VSP

PubMed Central

Kohout, Susy C.; Bell, Sarah C.; Liu, Lijun; Xu, Qiang; Minor, Daniel L.; Isacoff, Ehud Y.

2010-01-01

In the voltage sensing phosphatase, Ci-VSP, a voltage sensing domain (VSD) controls a lipid phosphatase domain (PD). The mechanism by which the domains are allosterically coupled is not well understood. Using an in vivo assay, we find that the inter-domain linker that connects the VSD to the PD is essential for coupling the full-length protein. Biochemical assays show that the linker is also needed for activity in the isolated PD. We identify a late step of VSD motion in the full-length protein that depends on the linker. Strikingly, this VSD motion is found to require PI(4,5)P2, a substrate of Ci-VSP. These results suggest that the voltage-driven motion of the VSD turns the enzyme on by rearranging the linker into an activated conformation, and that this activated conformation is stabilized by PI(4,5)P2. We propose that Ci-VSP activity is self-limited because its decrease of PI(4,5)P2 levels decouples the VSD from the enzyme. PMID:20364128

A collaborative network middleware project by Lambda Station, TeraPaths, and Phoebus

NASA Astrophysics Data System (ADS)

Bobyshev, A.; Bradley, S.; Crawford, M.; DeMar, P.; Katramatos, D.; Shroff, K.; Swany, M.; Yu, D.

2010-04-01

The TeraPaths, Lambda Station, and Phoebus projects, funded by the US Department of Energy, have successfully developed network middleware services that establish on-demand and manage true end-to-end, Quality-of-Service (QoS) aware, virtual network paths across multiple administrative network domains, select network paths and gracefully reroute traffic over these dynamic paths, and streamline traffic between packet and circuit networks using transparent gateways. These services improve network QoS and performance for applications, playing a critical role in the effective use of emerging dynamic circuit network services. They provide interfaces to applications, such as dCache SRM, translate network service requests into network device configurations, and coordinate with each other to setup up end-to-end network paths. The End Site Control Plane Subsystem (ESCPS) builds upon the success of the three projects by combining their individual capabilities into the next generation of network middleware. ESCPS addresses challenges such as cross-domain control plane signalling and interoperability, authentication and authorization in a Grid environment, topology discovery, and dynamic status tracking. The new network middleware will take full advantage of the perfSONAR monitoring infrastructure and the Inter-Domain Control plane efforts and will be deployed and fully vetted in the Large Hadron Collider data movement environment.

Crystal structure of Src-like adaptor protein 2 reveals close association of SH3 and SH2 domains through β-sheet formation.

PubMed

Wybenga-Groot, Leanne E; McGlade, C Jane

2013-12-01

The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3-SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3-SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3-SH2 connector, and SH2 domain of SLAP2 (SLAP2-32). While both domains adopt typical folds, the short SH3-SH2 connector places them in close association. Strand βe of the SH3 domain interacts with strand βA of the SH2 domain, resulting in the formation of a continuous β sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding. © 2013.

Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

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

Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs

The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. colimore » α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.« less

Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection.

PubMed

Pernigo, Stefano; Fukuzawa, Atsushi; Beedle, Amy E M; Holt, Mark; Round, Adam; Pandini, Alessandro; Garcia-Manyes, Sergi; Gautel, Mathias; Steiner, Roberto A

2017-01-03

The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Solution NMR studies of Chlorella virus DNA ligase-adenylate.

PubMed

Piserchio, Andrea; Nair, Pravin A; Shuman, Stewart; Ghose, Ranajeet

2010-01-15

DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3'-OH/5'-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone (15)N spin relaxation and (15)N,(1)H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5'-phosphate. Copyright 2009 Elsevier Ltd. All rights reserved.

Crystal Structure of the FERM-SH2 Module of Human Jak2.

PubMed

McNally, Randall; Toms, Angela V; Eck, Michael J

2016-01-01

Jak-family tyrosine kinases mediate signaling from diverse cytokine receptors. Binding of Jaks to their cognate receptors is mediated by their N-terminal region, which contains FERM and SH2 domains. Here we describe the crystal structure of the FERM-SH2 region of Jak2 at 3.0Å resolution. The structure reveals that these domains and their flanking linker segments interact intimately to form an integrated structural module. The Jak2 FERM-SH2 structure closely resembles that recently described for Tyk2, another member of the Jak family. While the overall architecture and interdomain orientations are preserved between Jak2 and Tyk2, we identify residues in the putative receptor-binding groove that differ between the two and may contribute to the specificity of receptor recognition. Analysis of Jak mutations that are reported to disrupt receptor binding reveals that they lie in the hydrophobic core of the FERM domain, and are thus expected to compromise the structural integrity of the FERM-SH2 unit. Similarly, analysis of mutations in Jak3 that are associated with severe combined immunodeficiency suggests that they compromise Jak3 function by destabilizing the FERM-SH2 structure.

Insertion of inter-domain linkers improves expression and bioactivity of Zygote arrest (Zar) fusion proteins.

PubMed

Cook, Jonathan M; Charlesworth, Amanda

2017-04-01

Developmentally important proteins that are crucial for fertilization and embryogenesis are synthesized through highly regulated translation of maternal mRNA. The Zygote arrest proteins, Zar1 and Zar2, are crucial for embryogenesis and have been implicated in binding mRNA and repressing mRNA translation. To investigate Zar1 and Zar2, the full-length proteins had been fused to glutathione-S-transferase (GST) or MS2 protein tags with minimal inter-domain linkers derived from multiple cloning sites; however, these fusion proteins expressed poorly and/or lacked robust function. Here, we tested the effect of inserting additional linkers between the fusion domains. Three linkers were tested, each 17 amino acids long with different physical and chemical properties: flexible hydrophilic, rigid extended or rigid helical. In the presence of any of the three linkers, GST-Zar1 and GST-Zar2 had fewer breakdown products. Moreover, in the presence of any of the linkers, MS2-Zar1 was expressed to higher levels, and in dual luciferase tethered assays, both MS2-Zar1 and MS2-Zar2 repressed luciferase translation to a greater extent. These data suggest that for Zar fusion proteins, increasing the length of linkers, regardless of their physical or chemical properties, improves stability, expression and bioactivity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Anatomy of triply-periodic network assemblies: characterizing skeletal and inter-domain surface geometry of block copolymer gyroids.

PubMed

Prasad, Ishan; Jinnai, Hiroshi; Ho, Rong-Ming; Thomas, Edwin L; Grason, Gregory M

2018-05-09

Triply-periodic networks (TPNs), like the well-known gyroid and diamond network phases, abound in soft matter assemblies, from block copolymers (BCPs), lyotropic liquid crystals and surfactants to functional architectures in biology. While TPNs are, in reality, volume-filling patterns of spatially-varying molecular composition, physical and structural models most often reduce their structure to lower-dimensional geometric objects: the 2D interfaces between chemical domains; and the 1D skeletons that thread through inter-connected, tubular domains. These lower-dimensional structures provide a useful basis of comparison to idealized geometries based on triply-periodic minimal, or constant-mean curvature surfaces, and shed important light on the spatially heterogeneous packing of molecular constituents that form the networks. Here, we propose a simple, efficient and flexible method to extract a 1D skeleton from 3D volume composition data of self-assembled networks. We apply this method to both self-consistent field theory predictions as well as experimental electron microtomography reconstructions of the double-gyroid phase of an ABA triblock copolymer. We further demonstrate how the analysis of 1D skeleton, 2D inter-domain surfaces, and combinations therefore, provide physical and structural insight into TPNs, across multiple length scales. Specifically, we propose and compare simple measures of network chirality as well as domain thickness, and analyze their spatial and statistical distributions in both ideal (theoretical) and non-ideal (experimental) double gyroid assemblies.

Structural basis for the glucan phosphatase activity of Starch Excess4

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

Vander Kooi, Craig W.; Taylor, Adam O.; Pace, Rachel M.

Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structuremore » of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydrate-binding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.« less

Structural modeling of the AhR:ARNT complex in the bHLH-PASA-PASB region elucidates the key determinants of dimerization.

PubMed

Corrada, Dario; Denison, Michael S; Bonati, Laura

2017-05-02

Elucidation of the dimerization process of the aryl hydrocarbon receptor (AhR) with the AhR nuclear translocator (ARNT) is crucial for understanding the mechanisms underlying the functional activity of AhR, including mediation of the toxicity of environmental contaminants. In this work, for the first time a structural model of the AhR:ARNT dimer encompassing the entire bHLH-PASA-PASB domain region is proposed. It is developed by using a template-based modeling approach, relying on the recently available crystallographic structures of two dimers of homologous systems in the bHLH-PAS family of proteins: the CLOCK:BMAL1 and the HIF2α:ARNT heterodimers. The structural and energetic characteristics of the modeled AhR:ARNT protein-protein interface are determined by evaluating the variations in solvent accessible surface area, the total binding free energy and the per-residue free energy contributions obtained by the MM-GBSA method and the Energy Decomposition Analysis. The analyses of the intricate network of inter-domain interactions at the dimerization interfaces provide insights into the key determinants of dimerization. These are confirmed by comparison of the computational findings with the available experimental mutagenesis and functional analysis data. The results presented here on the AhR:ARNT dimer structure and interactions provide a framework to start analyzing the mechanism of AhR transformation into its functional DNA binding form.

Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme.

PubMed

Stogios, Peter J; Shakya, Tushar; Evdokimova, Elena; Savchenko, Alexei; Wright, Gerard D

2011-01-21

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.

Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity.

PubMed

Chong, P Andrew; Lin, Hong; Wrana, Jeffrey L; Forman-Kay, Julie D

2010-10-26

Smad ubiquitination regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that participates in degradation of TGF-β receptors and other targets. Smurf2 WW domains recognize PPXY (PY) motifs on ubiquitin ligase target proteins or on adapters, such as Smad7, that bind to E3 target proteins. We previously demonstrated that the isolated WW3 domain of Smurf2, but not the WW2 domain, can directly bind to a Smad7 PY motif. We show here that the WW2 augments this interaction by binding to the WW3 and making auxiliary contacts with the PY motif and a novel E/D-S/T-P motif, which is N-terminal to all Smad PY motifs. The WW2 likely enhances the selectivity of Smurf2 for the Smad proteins. NMR titrations confirm that Smad1 and Smad2 are bound by Smurf2 with the same coupled WW domain arrangement used to bind Smad7. The analogous WW domains in the short isoform of Smurf1 recognize the Smad7 PY peptide using the same coupled mechanism. However, a longer Smurf1 isoform, which has an additional 26 residues in the inter-WW domain linker, is only partially able to use the coupled WW domain binding mechanism. The longer linker results in a decrease in affinity for the Smad7 peptide. Interdomain coupling of WW domains enhances selectivity and enables the tuning of interactions by isoform switching.

Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity

PubMed Central

Chong, P. Andrew; Lin, Hong; Wrana, Jeffrey L.; Forman-Kay, Julie D.

2010-01-01

Smad ubiquitination regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that participates in degradation of TGF-β receptors and other targets. Smurf2 WW domains recognize PPXY (PY) motifs on ubiquitin ligase target proteins or on adapters, such as Smad7, that bind to E3 target proteins. We previously demonstrated that the isolated WW3 domain of Smurf2, but not the WW2 domain, can directly bind to a Smad7 PY motif. We show here that the WW2 augments this interaction by binding to the WW3 and making auxiliary contacts with the PY motif and a novel E/D-S/T-P motif, which is N-terminal to all Smad PY motifs. The WW2 likely enhances the selectivity of Smurf2 for the Smad proteins. NMR titrations confirm that Smad1 and Smad2 are bound by Smurf2 with the same coupled WW domain arrangement used to bind Smad7. The analogous WW domains in the short isoform of Smurf1 recognize the Smad7 PY peptide using the same coupled mechanism. However, a longer Smurf1 isoform, which has an additional 26 residues in the inter-WW domain linker, is only partially able to use the coupled WW domain binding mechanism. The longer linker results in a decrease in affinity for the Smad7 peptide. Interdomain coupling of WW domains enhances selectivity and enables the tuning of interactions by isoform switching. PMID:20937913

Crystal structure of the solute-binding protein BxlE from Streptomyces thermoviolaceus OPC-520 complexed with xylobiose.

PubMed

Tomoo, Koji; Miki, Yasuhiro; Morioka, Hideaki; Seike, Kiho; Ishida, Toshimasa; Ikenishi, Sadao; Miyamoto, Katsushiro; Hasegawa, Tomokazu; Yamano, Akihito; Hamada, Kensaku; Tsujibo, Hiroshi

2017-06-01

BxlE from Streptomyces thermoviolaceus OPC-520 is a xylo-oligosaccharide (mainly xylobiose)-binding protein that serves as the initial receptor for the bacterial ABC-type xylo-oligosaccharide transport system. To determine the ligand-binding mechanism of BxlE, X-ray structures of ligand-free (open form) and ligand (xylobiose)-bound (closed form) BxlE were determined at 1.85 Å resolution. BxlE consists of two globular domains that are linked by two β-strands, with the cleft at the interface of the two domains creating the ligand-binding pocket. In the ligand-free open form, this pocket consists of a U-shaped and negatively charged groove located between the two domains. In the xylobiose-bound closed form of BxlE, both the N and C domains move to fold the ligand without conformational changes in either domain. Xylobiose is buried in the groove and wrapped by the N-domain mainly via hydrogen bond interactions and by the C-domain primarily via non-polar interactions with Trp side chains. In addition to the concave shape matching the binding of xylobiose, an inter-domain salt bridge between Asp-47 and Lys-294 limits the space in the ligand-binding site. This domain-stabilized mechanism of ligand binding to BxlE is a unique feature that is not observed with other solute-binding proteins. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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

Stogios, Peter J.; Shakya, Tushar; Evdokimova, Elena

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 {angstrom} resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity,more » indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2{double_prime}) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.« less

Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

PubMed

Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

2016-10-01

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

Identification of structural domains in proteins by a graph heuristic.

PubMed

Wernisch, L; Hunting, M; Wodak, S J

1999-05-15

A novel automatic procedure for identifying domains from protein atomic coordinates is presented. The procedure, termed STRUDL (STRUctural Domain Limits), does not take into account information on secondary structures and handles any number of domains made up of contiguous or non-contiguous chain segments. The core algorithm uses the Kernighan-Lin graph heuristic to partition the protein into residue sets which display minimum interactions between them. These interactions are deduced from the weighted Voronoi diagram. The generated partitions are accepted or rejected on the basis of optimized criteria, representing basic expected physical properties of structural domains. The graph heuristic approach is shown to be very effective, it approximates closely the exact solution provided by a branch and bound algorithm for a number of test proteins. In addition, the overall performance of STRUDL is assessed on a set of 787 representative proteins from the Protein Data Bank by comparison to domain definitions in the CATH protein classification. The domains assigned by STRUDL agree with the CATH assignments in at least 81% of the tested proteins. This result is comparable to that obtained previously using PUU (Holm and Sander, Proteins 1994;9:256-268), the only other available algorithm designed to identify domains with any number of non-contiguous chain segments. A detailed discussion of the structures for which our assignments differ from those in CATH brings to light some clear inconsistencies between the concept of structural domains based on minimizing inter-domain interactions and that of delimiting structural motifs that represent acceptable folding topologies or architectures. Considering both concepts as complementary and combining them in a layered approach might be the way forward.

Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates.

PubMed Central

Walter, N G; Burke, J M

1997-01-01

Current methods for evaluating the kinetics of ribozyme-catalyzed reactions rely primarily on the use of radiolabeled RNA substrates, and so require tedious electrophoretic separation and quantitation of reaction products for each data point in any experiment. Here, we report the use of fluorescent substrates for real-time analysis of the time course of reactions of the hairpin ribozyme. Fluorescence of 3' fluorescein-labeled substrates was quenched upon binding to the hairpin ribozyme or its isolated substrate-binding strand (SBS), under conditions of ribozyme or SBS excess. This decrease was accompanied by an increase in anisotropy, and resulted from a base-specific quenching by a guanosine residue added to the 5' end of the SBS, close to fluorescein in the complex. Fluorescence quenching was used to determine rate constants for substrate binding (1.4 x 10(8) M(-1) min(-1)), cleavage (0.15 min(-1)), and substrate dissociation (0.010 min(-1)) by a structurally well-defined ribozyme at 25 degrees C in 50 mM Tris-HCI, pH 7.5, 12 mM MgCl2. These rates are in excellent agreement with those obtained using traditional radioisotopic methods. Measurements of dissociation rates provided physical support for interdomain interactions within the substrate-ribozyme complex. We estimate that 2.1 kcal/mol of additional substrate binding energy is provided by the B domain of the ribozyme. Part of this free energy apparently stems from coaxial stacking of helices in the hinge region between domains, and it is plausible that the remainder might be contributed by direct interactions with loop B. The fluorescence quenching and dequenching methods described here should be readily adaptable to studying a wide variety of RNA interactions and reactions using ribozymes and other model systems. PMID:9085846

Cross-talk between the ligand- and DNA-binding domains of estrogen receptor.

PubMed

Huang, Wei; Greene, Geoffrey L; Ravikumar, Krishnakumar M; Yang, Sichun

2013-11-01

Estrogen receptor alpha (ERα) is a hormone-responsive transcription factor that contains several discrete functional domains, including a ligand-binding domain (LBD) and a DNA-binding domain (DBD). Despite a wealth of knowledge about the behaviors of individual domains, the molecular mechanisms of cross-talk between LBD and DBD during signal transduction from hormone to DNA-binding of ERα remain elusive. Here, we apply a multiscale approach combining coarse-grained (CG) and atomistically detailed simulations to characterize this cross-talk mechanism via an investigation of the ERα conformational landscape. First, a CG model of ERα is built based on crystal structures of individual LBDs and DBDs, with more emphasis on their interdomain interactions. Second, molecular dynamics simulations are implemented and enhanced sampling is achieved via the "push-pull-release" strategy in the search for different LBD-DBD orientations. Third, multiple energetically stable ERα conformations are identified on the landscape. A key finding is that estradiol-bound LBDs utilize the well-described activation helix H12 to pack and stabilize LBD-DBD interactions. Our results suggest that the estradiol-bound LBDs can serve as a scaffold to position and stabilize the DBD-DNA complex, consistent with experimental observations of enhanced DNA binding with the LBD. Final assessment using atomic-level simulations shows that these CG-predicted models are significantly stable within a 15-ns simulation window and that specific pairs of lysine residues in close proximity at the domain interfaces could serve as candidate sites for chemical cross-linking studies. Together, these simulation results provide a molecular view of the role of ERα domain interactions in response to hormone binding. Copyright © 2013 Wiley Periodicals, Inc.

Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer

PubMed Central

Siddiqui, Imtiaz A.; Asim, Mohammad; Hafeez, Bilal B.; Adhami, Vaqar M.; Tarapore, Rohinton S.; Mukhtar, Hasan

2011-01-01

Androgen deprivation therapy is the major treatment for advanced prostate cancer (PCa). However, it is a temporary remission, and the patients almost inevitably develop hormone refractory prostate cancer (HRPC). HRPC is almost incurable, although most HRPC cells still express androgen receptor (AR) and depend on the AR for growth, making AR a prime drug target. Here, we provide evidence that epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is a direct antagonist of androgen action. In silico modeling and FRET-based competition assay showed that EGCG physically interacts with the ligand-binding domain of AR by replacing a high-affinity labeled ligand (IC50 0.4 μM). The functional consequence of this interaction was a decrease in AR-mediated transcriptional activation, which was due to EGCG mediated inhibition of interdomain N-C termini interaction of AR. Treatment with EGCG also repressed the transcriptional activation by a hotspot mutant AR (T877A) expressed ectopically as well as the endogenous AR mutant. As the physiological consequence of AR antagonism, EGCG repressed R1881-induced PCa cell growth. In a xenograft model, EGCG was found to inhibit AR nuclear translocation and protein expression. We also observed a significant down-regulation of androgen-regulated miRNA-21 and up-regulation of a tumor suppressor, miRNA-330, in tumors of mice treated with EGCG. Taken together, we provide evidence that EGCG functionally antagonizes androgen action at multiple levels, resulting in inhibition of PCa growth.—Siddiqui, I. A., Asim, M., Hafeez, B. B., Adhami, V. M., Tarapore, R. S., Mukhtar, H. Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer. PMID:21177307

Mechanism of MicroRNA-Target Interaction: Molecular Dynamics Simulations and Thermodynamics Analysis

PubMed Central

Wang, Yonghua; Li, Yan; Ma, Zhi; Yang, Wei; Ai, Chunzhi

2010-01-01

MicroRNAs (miRNAs) are endogenously produced ∼21-nt riboregulators that associate with Argonaute (Ago) proteins to direct mRNA cleavage or repress the translation of complementary RNAs. Capturing the molecular mechanisms of miRNA interacting with its target will not only reinforce the understanding of underlying RNA interference but also fuel the design of more effective small-interfering RNA strands. To address this, in the present work the RNA-bound (Ago-miRNA, Ago-miRNA-target) and RNA-free Ago forms were analyzed by performing both molecular dynamics simulations and thermodynamic analysis. Based on the principal component analysis results of the simulation trajectories as well as the correlation analysis in fluctuations of residues, we discover that: 1) three important (PAZ, Mid and PIWI) domains exist in Argonaute which define the global dynamics of the protein; 2) the interdomain correlated movements are so crucial for the interaction of Ago-RNAs that they not only facilitate the relaxation of the interactions between residues surrounding the RNA binding channel but also induce certain conformational changes; and 3) it is just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release. In addition, by thermodynamic analysis we also discover that for both the guide RNA 5′-end recognition and the facilitated site-specific cleavage of the target, the presence of two metal ions (of Mg2+) plays a predominant role, and this conclusion is consistent with the observed enzyme catalytic cleavage activity in the ternary complex (Ago-miRNA-mRNA). Our results find that it is the set of arginine amino acids concentrated in the nucleotide-binding channel in Ago, instead of the conventionally-deemed seed base-paring, that makes greater contributions in stabilizing the binding of the nucleic acids to Ago. PMID:20686687

NH2-terminal sequence truncation decreases the stability of bovine rhodanese, minimally perturbs its crystal structure, and enhances interaction with GroEL under native conditions.

PubMed

Trevino, R J; Gliubich, F; Berni, R; Cianci, M; Chirgwin, J M; Zanotti, G; Horowitz, P M

1999-05-14

The NH2-terminal sequence of rhodanese influences many of its properties, ranging from mitochondrial import to folding. Rhodanese truncated by >9 residues is degraded in Escherichia coli. Mutant enzymes with lesser truncations are recoverable and active, but they show altered active site reactivities (Trevino, R. J., Tsalkova, T., Dramer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P. M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-terminal sequence stabilizes the overall structure. We tested aspects of the conformations of these shortened species. Intrinsic and probe fluorescence showed that truncation decreased stability and increased hydrophobic exposure, while near UV CD suggested altered tertiary structure. Under native conditions, truncated rhodanese bound to GroEL and was released and reactivated by adding ATP and GroES, suggesting equilibrium between native and non-native conformers. Furthermore, GroEL assisted folding of denatured mutants to the same extent as wild type, although at a reduced rate. X-ray crystallography showed that Delta1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-terminal residues that contribute to global stability of the native structure in solution do not significantly alter contacts at the atomic level of the crystallized protein. The two-domain structure of rhodanese was not significantly altered by drastically different crystallization conditions or crystal packing suggesting rigidity of the native rhodanese domains and the stabilization of the interdomain interactions by the crystal environment. The results support a model in which loss of interactions near the rhodanese NH2 terminus does not distort the folded native structure but does facilitate the transition in solution to a molten globule state, which among other things, can interact with molecular chaperones.

Characterization of potential antiviral resistance mutations in hepatitis B virus reverse transcriptase sequences in treatment-naïve Chinese patients.

PubMed

Liu, Bao-Ming; Li, Tong; Xu, Jie; Li, Xiao-Guang; Dong, Jian-Ping; Yan, Ping; Yang, Jing-Xian; Yan, Ling; Gao, Zhi-Yong; Li, Wen-Peng; Sun, Xie-Wen; Wang, Yu-Hua; Jiao, Xiu-Juan; Hou, Chun-Sheng; Zhuang, Hui

2010-03-01

Full-length hepatitis B virus (HBV) reverse transcriptase (RT) sequences were amplified and sequenced among 192 nucleos(t)ide analogue (NA)-naïve Chinese patients with chronic hepatitis B. Deduced amino acids (AAs) at 42 previously reported potential NA resistance (NAr) mutation positions in RT region were analyzed. Patients were found with either B-genotype (28.65%) or C-genotype (71.35%) infections. Rt53, rt91, rt124, rt134, rt221, rt224, rt238 and rt256 were identified as B- and C-genotype-dependent polymorphic AA positions. AA substitutions at 11 classical NAr mutation positions, i.e. rt80, rt169, rt173, rt180, rt181, rt184, rt194, rt202, rt204, rt236 and rt250, were not detected. However, potential NAr mutations were found in 30.73% (59/192) isolates, which involved 18 positions including rt53, rt207, rt229, rt238 and rt256, etc. The concomitant AA changes of HBsAg occurred in 16.67% (32/192) isolates including sG145R mutation. One-third of mutation positions were located in functional RT domains (e.g. rt207 and rt233), A-B interdomains (overlapping HBsAg 'a' determinant and showing most concomitant immune-associated mutations) and non-A-B interdomains (e.g. rt191 and rt213), respectively. Genotypes B and C each showed several preferred positions to mutate. These results might provide insights into understanding the evolution and selection basis of NAr HBV strains under antiviral therapy.

Hybrid protection algorithms based on game theory in multi-domain optical networks

NASA Astrophysics Data System (ADS)

Guo, Lei; Wu, Jingjing; Hou, Weigang; Liu, Yejun; Zhang, Lincong; Li, Hongming

2011-12-01

With the network size increasing, the optical backbone is divided into multiple domains and each domain has its own network operator and management policy. At the same time, the failures in optical network may lead to a huge data loss since each wavelength carries a lot of traffic. Therefore, the survivability in multi-domain optical network is very important. However, existing survivable algorithms can achieve only the unilateral optimization for profit of either users or network operators. Then, they cannot well find the double-win optimal solution with considering economic factors for both users and network operators. Thus, in this paper we develop the multi-domain network model with involving multiple Quality of Service (QoS) parameters. After presenting the link evaluation approach based on fuzzy mathematics, we propose the game model to find the optimal solution to maximize the user's utility, the network operator's utility, and the joint utility of user and network operator. Since the problem of finding double-win optimal solution is NP-complete, we propose two new hybrid protection algorithms, Intra-domain Sub-path Protection (ISP) algorithm and Inter-domain End-to-end Protection (IEP) algorithm. In ISP and IEP, the hybrid protection means that the intelligent algorithm based on Bacterial Colony Optimization (BCO) and the heuristic algorithm are used to solve the survivability in intra-domain routing and inter-domain routing, respectively. Simulation results show that ISP and IEP have the similar comprehensive utility. In addition, ISP has better resource utilization efficiency, lower blocking probability, and higher network operator's utility, while IEP has better user's utility.

Computational Architecture of the Parieto-Frontal Network Underlying Cognitive-Motor Control in Monkeys

PubMed Central

Borra, Elena; Visco-Comandini, Federica; Averbeck, Bruno B.

2017-01-01

The statistical structure of intrinsic parietal and parieto-frontal connectivity in monkeys was studied through hierarchical cluster analysis. Based on their inputs, parietal and frontal areas were grouped into different clusters, including a variable number of areas that in most instances occupied contiguous architectonic fields. Connectivity tended to be stronger locally: that is, within areas of the same cluster. Distant frontal and parietal areas were targeted through connections that in most instances were reciprocal and often of different strength. These connections linked parietal and frontal clusters formed by areas sharing basic functional properties. This led to five different medio-laterally oriented pillar domains spanning the entire extent of the parieto-frontal system, in the posterior parietal, anterior parietal, cingulate, frontal, and prefrontal cortex. Different information processing streams could be identified thanks to inter-domain connectivity. These streams encode fast hand reaching and its control, complex visuomotor action spaces, hand grasping, action/intention recognition, oculomotor intention and visual attention, behavioral goals and strategies, and reward and decision value outcome. Most of these streams converge on the cingulate domain, the main hub of the system. All of them are embedded within a larger eye–hand coordination network, from which they can be selectively set in motion by task demands. PMID:28275714

Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

PubMed

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

2009-01-01

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

Conformational Flexibility of Human Casein Kinase Catalytic Subunit Explored by Metadynamics

PubMed Central

Gouron, Aurélie; Milet, Anne; Jamet, Helene

2014-01-01

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation. PMID:24606937

Dissecting the protein architecture of DNA-binding transcription factors in bacteria and archaea.

PubMed

Rivera-Gómez, Nancy; Martínez-Núñez, Mario Alberto; Pastor, Nina; Rodriguez-Vazquez, Katya; Perez-Rueda, Ernesto

2017-08-01

Gene regulation at the transcriptional level is a central process in all organisms where DNA-binding transcription factors play a fundamental role. This class of proteins binds specifically at DNA sequences, activating or repressing gene expression as a function of the cell's metabolic status, operator context and ligand-binding status, among other factors, through the DNA-binding domain (DBD). In addition, TFs may contain partner domains (PaDos), which are involved in ligand binding and protein-protein interactions. In this work, we systematically evaluated the distribution, abundance and domain organization of DNA-binding TFs in 799 non-redundant bacterial and archaeal genomes. We found that the distributions of the DBDs and their corresponding PaDos correlated with the size of the genome. We also identified specific combinations between the DBDs and their corresponding PaDos. Within each class of DBDs there are differences in the actual angle formed at the dimerization interface, responding to the presence/absence of ligands and/or crystallization conditions, setting the orientation of the resulting helices and wings facing the DNA. Our results highlight the importance of PaDos as central elements that enhance the diversity of regulatory functions in all bacterial and archaeal organisms, and our results also demonstrate the role of PaDos in sensing diverse signal compounds. The highly specific interactions between DBDs and PaDos observed in this work, together with our structural analysis highlighting the difficulty in predicting both inter-domain geometry and quaternary structure, suggest that these systems appeared once and evolved with diverse duplication events in all the analysed organisms.

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1

PubMed Central

Ren, Hong Yu; Grove, Diane E.; De La Rosa, Oxana; Houck, Scott A.; Sopha, Pattarawut; Van Goor, Fredrick; Hoffman, Beth J.; Cyr, Douglas M.

2013-01-01

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR. PMID:23924900

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.

PubMed

Ren, Hong Yu; Grove, Diane E; De La Rosa, Oxana; Houck, Scott A; Sopha, Pattarawut; Van Goor, Fredrick; Hoffman, Beth J; Cyr, Douglas M

2013-10-01

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.

Impact of disease-causing mutations on inter-domain interactions in cMyBP-C: a steered molecular dynamics study.

PubMed

Krishnamoorthy, Navaneethakrishnan; Gajendrarao, Poornima; Olivotto, Iacopo; Yacoub, Magdi

2017-07-01

The molecular interactions of the sarcomeric proteins are essential in the regulation of various cardiac functions. Mutations in the gene MYBPC3 coding for cardiac myosin-binding protein-C (cMyBP-C), a multi-domain protein, are the most common cause of hypertrophic cardiomyopathy (HCM). The N-terminal complex, C1-motif-C2 is a central region in cMyBP-C for the regulation of cardiac muscle contraction. However, the mechanism of binding/unbinding of this complex during health and disease is unknown. Here, we study possible mechanisms of unbinding using steered molecular dynamics simulations for the complex in the wild type, in single mutations (E258K in C1, E441K in C2), as well as in a double mutation (E258K in C1 + E441K in C2), which are associated with severe HCM. The observed molecular events and the calculation of force utilized for the unbinding suggest the following: (i) double mutation can encourage the formation of rigid complex that required large amount of force and long-time to unbind, (ii) C1 appears to start to unbind ahead of C2 regardless of the mutation, and (iii) unbinding of C2 requires larger amount of force than C1. This molecular insight suggests that key HCM-causing mutations might significantly modify the native affinity required for the assembly of the domains in cMyBP-C, which is essential for normal cardiac function.

Mechanisms of Intramolecular Communication in a Hyperthermophilic Acylaminoacyl Peptidase: A Molecular Dynamics Investigation

PubMed Central

Papaleo, Elena; Renzetti, Giulia; Tiberti, Matteo

2012-01-01

Protein dynamics and the underlying networks of intramolecular interactions and communicating residues within the three-dimensional (3D) structure are known to influence protein function and stability, as well as to modulate conformational changes and allostery. Acylaminoacyl peptidase (AAP) subfamily of enzymes belongs to a unique class of serine proteases, the prolyl oligopeptidase (POP) family, which has not been thoroughly investigated yet. POPs have a characteristic multidomain three-dimensional architecture with the active site at the interface of the C-terminal catalytic domain and a β-propeller domain, whose N-terminal region acts as a bridge to the hydrolase domain. In the present contribution, protein dynamics signatures of a hyperthermophilic acylaminoacyl peptidase (AAP) of the prolyl oligopeptidase (POP) family, as well as of a deletion variant and alanine mutants (I12A, V13A, V16A, L19A, I20A) are reported. In particular, we aimed at identifying crucial residues for long range communications to the catalytic site or promoting the conformational changes to switch from closed to open ApAAP conformations. Our investigation shows that the N-terminal α1-helix mediates structural intramolecular communication to the catalytic site, concurring to the maintenance of a proper functional architecture of the catalytic triad. Main determinants of the effects induced by α1-helix are a subset of hydrophobic residues (V16, L19 and I20). Moreover, a subset of residues characterized by relevant interaction networks or coupled motions have been identified, which are likely to modulate the conformational properties at the interdomain interface. PMID:22558199

Molecular Insights into the Potential Insecticidal Interaction of β-Dihydroagarofuran Derivatives with the H Subunit of V-ATPase.

PubMed

Wei, Jielu; Li, Ding; Xi, Xin; Liu, Lulu; Zhao, Ximei; Wu, Wenjun; Zhang, Jiwen

2017-10-11

Celangulin V (CV), one of dihydroagarofuran sesquiterpene polyesters isolated from Chinese bittersweet ( Celastrus angulatus Maxim), is famous natural botanical insecticide. Decades of research suggests that is displays excellent insecticidal activity against some insects, such as Mythimna separata Walker. Recently, it has been validated that the H subunit of V-ATPase is one of the target proteins of the insecticidal dihydroagarofuran sesquiterpene polyesters. As a continuation of the development of new pesticides from these natural products, a series of β-dihydroagarofuran derivatives have been designed and synthesized. The compound JW-3, an insecticidal derivative of CV with a p -fluorobenzyl group, exhibits higher insecticidal activity than CV. In this study, the potential inhibitory effect aused by the interaction of JW-3 with the H subunit of V-ATPase c was verified by confirmatory experiments at the molecular level. Both spectroscopic techniques and isothermal titration calorimetry measurements showed the binding of JW-3 to the subunit H of V-ATPase was specific and spontaneous. In addition, the possible mechanism of action of the compound was discussed. Docking results indicated compound JW-3 could bind well in 'the interdomain cleft' of the V-ATPase subunit H by the hydrogen bonding and make conformation of the ligand-protein complex become more stable. All results are the further validations of the hypothesis, that the target protein of insecticidal dihydroagarofuran sesquiterpene polyesters and their β-dihydroagarofuran derivatives is the subunit H of V-ATPase. The results also provide new ideas for developing pesticides acting on V-ATPase of insects.

Multinational Experiment 7. Outcome 4: Methodology to Understand Inter-Domain Dependencies and Vulnerabilities Guide v. 1.0

DTIC Science & Technology

2013-01-31

the methodology had reached its full maturity and proposed a number of changes, mainly pertaining to its clarification. Recommendations stemming from...34’ "’ ’iii ao > c:iij ·- c: N 8: c( "’ "’ .!! ::?i Qj ::::s "’ ’u ~ c. c: O :c~ l£ c: ~ .Q 8. M Cll ::::J ~ 0 ~ ... Qj ..!!!c ~9 c...example, the analyst should be able to superimpose or switch from the C2 Kmap to the intelligence Kmap when he or she works on the dependencies stemming

Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes.

PubMed

Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

2016-10-18

The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains-In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

Reciprocal Influence of Protein Domains in the Cold-Adapted Acyl Aminoacyl Peptidase from Sporosarcina psychrophila

PubMed Central

Parravicini, Federica; Natalello, Antonino; Papaleo, Elena; De Gioia, Luca; Doglia, Silvia Maria; Lotti, Marina; Brocca, Stefania

2013-01-01

Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/β-hydrolase domain and by an N-terminal β-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the β-propeller. The α1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated β-propeller. A single charged residue (E10) in the α1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution. PMID:23457536

Reciprocal influence of protein domains in the cold-adapted acyl aminoacyl peptidase from Sporosarcina psychrophila.

PubMed

Parravicini, Federica; Natalello, Antonino; Papaleo, Elena; De Gioia, Luca; Doglia, Silvia Maria; Lotti, Marina; Brocca, Stefania

2013-01-01

Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/β-hydrolase domain and by an N-terminal β-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the β-propeller. The α1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated β-propeller. A single charged residue (E10) in the α1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution.

DevS Oxy Complex Stability Identifies this Heme Protein as a Gas Sensor in Mycobacterium tuberculosis Dormancy†

PubMed Central

Ioanoviciu, Alexandra; Meharenna, Yergalem T.; Poulos, Thomas L.; Ortiz de Montellano, Paul R.

2009-01-01

DevS is one of the two sensing kinases responsible for DevR activation and the subsequent entry of Mycobacterium tuberculosis into dormancy. Full length wild-type DevS forms a stable oxy-ferrous complex. The DevS autooxidation rates are extremely low (half-lives > 24 h) in the presence of cations such as K+, Na+, Mg2+, and Ca2+. At relatively high concentrations (100 µM), Fe3+ mildly increases the autooxidation rate (six-fold increase) while Cu2+ accelerates autooxidation more than 1500-fold. Contrary to expectations, removal of the key hydrogen bond between the iron-coordinated oxygen and Tyr171 in the Y171F mutant provides a protein of comparable stability to autooxidation and similar oxygen dissociation rate. This correlates with our earlier finding that the Y171F mutant and wild-type kinase activities are similarly regulated by the binding of oxygen: namely, the ferrous 5c complex is active whereas the oxy ferrous 6c species is inactive. Our results indicate that DevS is a gas sensor in vivo rather than a redox sensor and that the stability of its ferrous-oxy complex is enhanced by inter-domain interactions. PMID:19463006

Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid

PubMed Central

Dutta, Moumita; Pollard, Dominic J.; Goldstone, David C.; Ramos, Andres; Müllers, Erik; Stirnnagel, Kristin; Stanke, Nicole; Lindemann, Dirk; Taylor, William R.; Rosenthal, Peter B.

2016-01-01

The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN—CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold. PMID:27829070

Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

PubMed

Tan, Cheng; Li, Wenfei; Wang, Wei

2013-12-19

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

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

Wang, Hui; Klein, Michael G.; Snell, Gyorgy

Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structuremore » reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.« less

Motion Tree Delineates Hierarchical Structure of Protein Dynamics Observed in Molecular Dynamics Simulation

PubMed Central

Moritsugu, Kei; Koike, Ryotaro; Yamada, Kouki; Kato, Hiroaki; Kidera, Akinori

2015-01-01

Molecular dynamics (MD) simulations of proteins provide important information to understand their functional mechanisms, which are, however, likely to be hidden behind their complicated motions with a wide range of spatial and temporal scales. A straightforward and intuitive analysis of protein dynamics observed in MD simulation trajectories is therefore of growing significance with the large increase in both the simulation time and system size. In this study, we propose a novel description of protein motions based on the hierarchical clustering of fluctuations in the inter-atomic distances calculated from an MD trajectory, which constructs a single tree diagram, named a “Motion Tree”, to determine a set of rigid-domain pairs hierarchically along with associated inter-domain fluctuations. The method was first applied to the MD trajectory of substrate-free adenylate kinase to clarify the usefulness of the Motion Tree, which illustrated a clear-cut dynamics picture of the inter-domain motions involving the ATP/AMP lid and the core domain together with the associated amplitudes and correlations. The comparison of two Motion Trees calculated from MD simulations of ligand-free and -bound glutamine binding proteins clarified changes in inherent dynamics upon ligand binding appeared in both large domains and a small loop that stabilized ligand molecule. Another application to a huge protein, a multidrug ATP binding cassette (ABC) transporter, captured significant increases of fluctuations upon binding a drug molecule observed in both large scale inter-subunit motions and a motion localized at a transmembrane helix, which may be a trigger to the subsequent structural change from inward-open to outward-open states to transport the drug molecule. These applications demonstrated the capabilities of Motion Trees to provide an at-a-glance view of various sizes of functional motions inherent in the complicated MD trajectory. PMID:26148295

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A.

PubMed

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-12-09

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities.

PubMed

Aydin, Cihan; Mukherjee, Sourav; Hanson, Alicia M; Frick, David N; Schiffer, Celia A

2013-12-01

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full-length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain-domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that the two domains of NS3/4A are dynamically coupled through the interface. Interestingly, mutations designed to disrupt this interface did not hinder the catalytic activities of either domain. In contrast, substrate cleavage and DNA unwinding by these mutants were mostly enhanced compared with the wild-type protein. Disrupting the interface did not significantly alter RNA unwinding activity; however, the full-length protein was more efficient in RNA unwinding than the isolated protease domain, suggesting a more direct role in RNA processing independent of the interface. Our findings suggest that HCV NS3/4A adopts an "extended" catalytically active conformation, and interface formation acts as a switch to regulate activity. We propose a unifying model connecting HCV NS3/4A conformational states and protease and helicase function, where interface formation and the dynamic interplay between the two enzymatic domains of HCV NS3/4A potentially modulate the protease and helicase activities in vivo. © 2013 The Protein Society.

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts*♦

PubMed Central

Barbieri, Christopher M.; Mack, Timothy R.; Robinson, Victoria L.; Miller, Matthew T.; Stock, Ann M.

2010-01-01

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo. PMID:20702407

Communication between Tandem cAMP Binding Domains in the Regulatory Subunit of Protein Kinase A-Iα as Revealed by Domain-silencing Mutations*

PubMed Central

McNicholl, E. Tyler; Das, Rahul; SilDas, Soumita; Taylor, Susan S.; Melacini, Giuseppe

2010-01-01

Protein kinase A (PKA) is the main receptor for the universal cAMP second messenger. PKA is a tetramer with two catalytic (C) and two regulatory (R) subunits, each including two tandem cAMP binding domains, i.e. CBD-A and -B. Structural investigations of RIα have revealed that although CBD-A plays a pivotal role in the cAMP-dependent inhibition of C, the main function of CBD-B is to regulate the access of cAMP to site A. To further understand the mechanism underlying the cross-talk between CBD-A and -B, we report here the NMR investigation of a construct of R, RIα-(119–379), which unlike previous fragments characterized by NMR, spans in full both CBDs. Our NMR studies were also extended to two mutants, R209K and the corresponding R333K, which severely reduce the affinity of cAMP for CBD-A and -B, respectively. The comparative NMR analysis of wild-type RIα-(119–379) and of the two domain silencing mutations has led to the definition at an unprecedented level of detail of both intra- and interdomain allosteric networks, revealing several striking differences between the two CBDs. First, the two domains, although homologous in sequence and structure, exhibit remarkably different responses to the R/K mutations especially at the β2-3 allosteric “hot spot.” Second, although the two CBDs are reciprocally coupled at the level of local unfolding of the hinge, the A-to-B and B-to-A pathways are dramatically asymmetrical at the level of global unfolding. Such an asymmetric interdomain cross-talk ensures efficiency and robustness in both the activation and de-activation of PKA. PMID:20202931

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A*

PubMed Central

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-01-01

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. PMID:27780868

AccR Is a Master Regulator Involved in Carbon Catabolite Repression of the Anaerobic Catabolism of Aromatic Compounds in Azoarcus sp. CIB*

PubMed Central

Valderrama, J. Andrés; Shingler, Victoria; Carmona, Manuel; Díaz, Eduardo

2014-01-01

Here we characterized the first known transcriptional regulator that accounts for carbon catabolite repression (CCR) control of the anaerobic catabolism of aromatic compounds in bacteria. The AccR response regulator of Azoarcus sp. CIB controls succinate-responsive CCR of the central pathways for the anaerobic catabolism of aromatics by this strain. Phosphorylation of AccR to AccR-P triggers a monomer-to-dimer transition as well as the ability to bind to the target promoter and causes repression both in vivo and in vitro. Substitution of the Asp60 phosphorylation target residue of the N-terminal receiver motif of AccR to a phosphomimic Glu residue generates a constitutively active derivative that behaves as a superrepressor of the target genes. AccR-P binds in vitro to a conserved inverted repeat (ATGCA-N6-TGCAT) present at two different locations within the PN promoter of the bzd genes for anaerobic benzoate degradation. Because the DNA binding-proficient C-terminal domain of AccR is monomeric, we propose an activation mechanism in which phosphorylation of Asp60 of AccR alleviates interdomain repression mediated by the N-terminal domain. The presence of AccR-like proteins encoded in the genomes of other β-proteobacteria of the Azoarcus/Thauera group further suggests that AccR constitutes a master regulator that controls anaerobic CCR in these bacteria. PMID:24302740

Agent-based model of fecal microbial transplant effect on bile acid metabolism on suppressing Clostridium difficile infection: an example of agent-based modeling of intestinal bacterial infection.

PubMed

Peer, Xavier; An, Gary

2014-10-01

Agent-based modeling is a computational modeling method that represents system-level behavior as arising from multiple interactions between the multiple components that make up a system. Biological systems are thus readily described using agent-based models (ABMs), as multi-cellular organisms can be viewed as populations of interacting cells, and microbial systems manifest as colonies of individual microbes. Intersections between these two domains underlie an increasing number of pathophysiological processes, and the intestinal tract represents one of the most significant locations for these inter-domain interactions, so much so that it can be considered an internal ecology of varying robustness and function. Intestinal infections represent significant disturbances of this internal ecology, and one of the most clinically relevant intestinal infections is Clostridium difficile infection (CDI). CDI is precipitated by the use of broad-spectrum antibiotics, involves the depletion of commensal microbiota, and alterations in bile acid composition in the intestinal lumen. We present an example ABM of CDI (the C. difficile Infection ABM, or CDIABM) to examine fundamental dynamics of the pathogenesis of CDI and its response to treatment with anti-CDI antibiotics and a newer treatment therapy, fecal microbial transplant. The CDIABM focuses on one specific mechanism of potential CDI suppression: commensal modulation of bile acid composition. Even given its abstraction, the CDIABM reproduces essential dynamics of CDI and its response to therapy, and identifies a paradoxical zone of behavior that provides insight into the role of intestinal nutritional status and the efficacy of anti-CDI therapies. It is hoped that this use case example of the CDIABM can demonstrate the usefulness of both agent-based modeling and the application of abstract functional representation as the biomedical community seeks to address the challenges of increasingly complex diseases with the goal of personalized medicine.

Agent-based model of Fecal Microbial Transplant effect on Bile Acid Metabolism on suppressing Clostridium difficile infection: an example of agent-based modeling of intestinal bacterial infection

PubMed Central

Peer, Xavier; An, Gary

2014-01-01

Agent-based modeling is a computational modeling method that represents system-level behavior as arising from multiple interactions between the multiple components that make up a system. Biological systems are thus readily described using agent-based models (ABMs), as multi-cellular organisms can be viewed as populations of interacting cells, and microbial systems manifest as colonies of individual microbes. Intersections between these two domains underlie an increasing number of pathophysiological processes, and the intestinal tract represents one of the most significant locations for these inter-domain interactions, so much so that it can be considered an internal ecology of varying robustness and function. Intestinal infections represent significant disturbances of this internal ecology, and one of the most clinically relevant intestinal infections is Clostridium difficile infection (CDI). CDI is precipitated by the use of broad-spectrum antibiotics, involves the depletion of commensal microbiota, and alterations in bile acid composition in the intestinal lumen. We present an example ABM of CDI (the Clostridium difficile Infection ABM, or CDIABM) to examine fundamental dynamics of the pathogenesis of CDI and its response to treatment with anti-CDI antibiotics and a newer treatment therapy, Fecal Microbial Transplant (FMT). The CDIABM focuses on one specific mechanism of potential CDI suppression: commensal modulation of bile acid composition. Even given its abstraction, the CDIABM reproduces essential dynamics of CDI and its response to therapy, and identifies a paradoxical zone of behavior that provides insight into the role of intestinal nutritional status and the efficacy of anti-CDI therapies. It is hoped that this use case example of the CDIABM can demonstrate the usefulness of both agent-based modeling and the application of abstract functional representation as the biomedical community seeks to address the challenges of increasingly complex diseases with the goal of personalized medicine. PMID:25168489

Structure of the glucagon receptor in complex with a glucagon analogue.

PubMed

Zhang, Haonan; Qiao, Anna; Yang, Linlin; Van Eps, Ned; Frederiksen, Klaus S; Yang, Dehua; Dai, Antao; Cai, Xiaoqing; Zhang, Hui; Yi, Cuiying; Cao, Can; He, Lingli; Yang, Huaiyu; Lau, Jesper; Ernst, Oliver P; Hanson, Michael A; Stevens, Raymond C; Wang, Ming-Wei; Reedtz-Runge, Steffen; Jiang, Hualiang; Zhao, Qiang; Wu, Beili

2018-01-03

Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR-NNC0640-mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation-which requires conformational changes of the stalk, first extracellular loop and TMD-that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.

Using THz Spectroscopy, Evolutionary Network Analysis Methods, and MD Simulation to Map the Evolution of Allosteric Communication Pathways in c-Type Lysozymes.

PubMed

Woods, Kristina N; Pfeffer, Juergen

2016-01-01

It is now widely accepted that protein function is intimately tied with the navigation of energy landscapes. In this framework, a protein sequence is not described by a distinct structure but rather by an ensemble of conformations. And it is through this ensemble that evolution is able to modify a protein's function by altering its landscape. Hence, the evolution of protein functions involves selective pressures that adjust the sampling of the conformational states. In this work, we focus on elucidating the evolutionary pathway that shaped the function of individual proteins that make-up the mammalian c-type lysozyme subfamily. Using both experimental and computational methods, we map out specific intermolecular interactions that direct the sampling of conformational states and accordingly, also underlie shifts in the landscape that are directly connected with the formation of novel protein functions. By contrasting three representative proteins in the family we identify molecular mechanisms that are associated with the selectivity of enhanced antimicrobial properties and consequently, divergent protein function. Namely, we link the extent of localized fluctuations involving the loop separating helices A and B with shifts in the equilibrium of the ensemble of conformational states that mediate interdomain coupling and concurrently moderate substrate binding affinity. This work reveals unique insights into the molecular level mechanisms that promote the progression of interactions that connect the immune response to infection with the nutritional properties of lactation, while also providing a deeper understanding about how evolving energy landscapes may define present-day protein function. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Critical Role of Interdomain Interactions in the Conformational Change and Catalytic Mechanism of Endoplasmic Reticulum Aminopeptidase 1.

PubMed

Stamogiannos, Athanasios; Maben, Zachary; Papakyriakou, Athanasios; Mpakali, Anastasia; Kokkala, Paraskevi; Georgiadis, Dimitris; Stern, Lawrence J; Stratikos, Efstratios

2017-03-14

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that is important for the generation of antigenic epitopes and major histocompatibility class I-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity, although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which C-terminal domain IV is either proximal or distal to active site domain II. To improve our understanding of the role of this conformational change in the catalytic mechanism of ERAP1, we used site-directed mutagenesis to perturb key salt bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce the catalytic efficiency and change the allosteric kinetic behavior. The variants were more efficiently activated by small peptides and bound a competitive inhibitor with weaker affinity and faster dissociation kinetics. Molecular dynamics analysis suggested that the mutations affect the conformational distribution of ERAP1, reducing the population of closed states. Small-angle X-ray scattering indicated that both the wild type and the ERAP1 variants are predominantly in an open conformational state in solution. Overall, our findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site. The extent of domain opening in ERAP1 probably underlies its specialization for antigenic peptide precursors and should be taken into account in inhibitor development efforts.

An Extended Structure of the APOBEC3G Catalytic Domain Suggests a Unique Holoenzyme Model

PubMed Central

Harjes, Elena; Gross, Phillip J.; Chen, Kuan-Ming; Lu, Yongjian; Shindo, Keisuke; Nowarski, Roni; Gross, John D.; Kotler, Moshe; Harris, Reuben S.; Matsuo, Hiroshi

2009-01-01

Summary Human APOBEC3G (A3G) belongs to a family of polynucleotide cytidine deaminases. This family includes APOBEC1 and AID, which edit APOB mRNA and antibody gene DNA, respectively. A3G deaminates cytidines to uridines in single-strand DNA and inhibits the replication of HIV-1, other retroviruses and retrotransposons. Although the mechanism of A3G-catalyzed DNA deamination has been investigated genetically and biochemically, atomic details are just starting to emerge. Here, we compare the DNA cytidine deaminase activities and NMR structures of two A3G catalytic domain constructs. The longer A3G191-384 protein is considerably more active than the shorter A3G198-384 variant. The longer structure has an α1 helix (residues 201–206) that was not apparent in the shorter protein and it contributes to catalytic activity through interactions with hydrophobic core structures (β1, β3, α5 and α6). Both A3G catalytic domain solution structures have a discontinuous β2 region that is clearly different than the continuous β2 strand of another family member APOBEC2. In addition, the longer A3G191-384 structure revealed part of the N-terminal pseudo-catalytic domain including the inter-domain linker and some of the last α-helix. These structured residues (191–196) enabled a novel full-length A3G model by providing physical overlap between the N-terminal pseudo-catalytic domain and the new C-terminal catalytic domain structure. Contrary to predictions, this structurally constrained model suggested that the two domains are tethered by structured residues and that the N- and C-terminal β2 regions are too distant from one another to participate in this interaction. PMID:19389408

Homology modeling and in silico prediction of Ulcerative colitis associated polymorphisms of NOD1.

PubMed

Majumdar, Ishani; Nagpal, Isha; Paul, Jaishree

2017-10-01

Cytosolic pattern recognition receptors play key roles in innate immune response. Nucleotide binding and oligomerisation domain containing protein 1 (NOD1) belonging to the Nod-like receptor C (NLRC) sub-family of Nod-like receptors (NLRs) is important for detection and clearance of intra-cellular Gram negative bacteria. NOD1 is involved in activation of pro-inflammatory pathways. Limited structural data is available for NOD1. Using different templates for each domain of NOD1, we determined the full-length homology model of NOD1. ADP binding amino acids within the nucleotide binding domain (NBD) of NOD1 were also predicted. Key residues in inter-domain interaction were identified by sequence comparison with Oryctolagus cuniculus NOD2, a related protein. Interactions between NBD and winged helix domain (WHD) were found to be conserved in NOD1. Functional and structural effect of single nucleotide polymorphisms within the NOD1 NBD domain associated with susceptibility risk to Ulcerative colitis (UC), an inflammatory disorder of the colon was evaluated by in silico studies. Mutations W219R and L349P were predicted to be damaging and disease associated by prediction programs SIFT, PolyPhen2, PANTHER, SNP&GO, PhD SNP and SNAP2. We further validated the effect of W219R and L349P mutation on NOD1 function in vitro. Elevated mRNA expression of pro-inflammatory cytokines IL8 and IL-1β was seen as compared to the wild type NOD1 in intestinal epithelial cell line HT29 when stimulated with NOD1 ligand. Thus, these mutations may indeed have a bearing on pathogenesis of inflammation during UC. Copyright © 2017 Elsevier Ltd. All rights reserved.

Unraveling the Solution-State Supramolecular Structures of Donor-Acceptor Polymers and their Influence on Solid-State Morphology and Charge-Transport Properties.

PubMed

Zheng, Yu-Qing; Yao, Ze-Fan; Lei, Ting; Dou, Jin-Hu; Yang, Chi-Yuan; Zou, Lin; Meng, Xiangyi; Ma, Wei; Wang, Jie-Yu; Pei, Jian

2017-11-01

Polymer self-assembly in solution prior to film fabrication makes solution-state structures critical for their solid-state packing and optoelectronic properties. However, unraveling the solution-state supramolecular structures is challenging, not to mention establishing a clear relationship between the solution-state structure and the charge-transport properties in field-effect transistors. Here, for the first time, it is revealed that the thin-film morphology of a conjugated polymer inherits the features of its solution-state supramolecular structures. A "solution-state supramolecular structure control" strategy is proposed to increase the electron mobility of a benzodifurandione-based oligo(p-phenylene vinylene) (BDOPV)-based polymer. It is shown that the solution-state structures of the BDOPV-based conjugated polymer can be tuned such that it forms a 1D rod-like structure in good solvent and a 2D lamellar structure in poor solvent. By tuning the solution-state structure, films with high crystallinity and good interdomain connectivity are obtained. The electron mobility significantly increases from the original value of 1.8 to 3.2 cm 2 V -1 s -1 . This work demonstrates that "solution-state supramolecular structure" control is critical for understanding and optimization of the thin-film morphology and charge-transport properties of conjugated polymers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Conformational flexibility of human casein kinase catalytic subunit explored by metadynamics.

PubMed

Gouron, Aurélie; Milet, Anne; Jamet, Helene

2014-03-04

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

Schürpf, Thomas; Chen, Qiang; Liu, Jin-huan

Developmental endothelial cell locus-1 (Del-1) glycoprotein is secreted by endothelial cells and a subset of macrophages. Del-1 plays a regulatory role in vascular remodeling and functions in innate immunity through interaction with integrin {alpha}{sub V}{beta}{sub 3}. Del-1 contains 3 epidermal growth factor (EGF)-like repeats and 2 discoidin-like domains. An Arg-Gly-Asp (RGD) motif in the second EGF domain (EGF2) mediates adhesion by endothelial cells and phagocytes. We report the crystal structure of its 3 EGF domains. The RGD motif of EGF2 forms a type II' {beta} turn at the tip of a long protruding loop, dubbed the RGD finger. Whereas EGF2more » and EGF3 constitute a rigid rod via an interdomain calcium ion binding site, the long linker between EGF1 and EGF2 lends considerable flexibility to EGF1. Two unique O-linked glycans and 1 N-linked glycan locate to the opposite side of EGF2 from the RGD motif. These structural features favor integrin binding of the RGD finger. Mutagenesis data confirm the importance of having the RGD motif at the tip of the RGD finger. A database search for EGF domain sequences shows that this RGD finger is likely an evolutionary insertion and unique to the EGF domain of Del-1 and its homologue milk fat globule-EGF 8. The RGD finger of Del-1 is a unique structural feature critical for integrin binding.« less

Molecular chirality and domain shapes in lipid monolayers on aqueous surfaces

NASA Astrophysics Data System (ADS)

Krüger, Peter; Lösche, Mathias

2000-11-01

The shapes of domain boundaries in the mesoscopic phase separation of phospholipids in aqueous surface monolayers are analyzed with particular attention to the influence of molecular chirality. We have calculated equilibrium shapes of such boundaries, and show that the concept of spontaneous curvature-derived from an effective pair potential between the chiral molecules-yields an adequate description of the contribution of chirality to the total energy of the system. For enantiomeric dipalmitoylphosphatidylcholine in pure monolayers, and in mixtures with impurities that adsorb preferentially at the (one-dimensional) boundary line between the isotropic and anisotropic fluid phases, such as cyanobiphenyl (5CB), a total energy term that includes line tension, electrostatic dipole-dipole interaction, and spontaneous curvature is sufficient to describe the shapes of well-separated domain boundaries in full detail. As soon as interdomain distances fall below the domain sizes upon compression of a monolayer, fluctuations take over in determining its detailed structural morphology. Using Minkowski measures for the well-studied dimyristoyl phosphatidic acid (DMPA)/cholesterol system, we show that calculations accounting for line tension, electrostatic repulsion, and molecular chirality yield boundary shapes that are of the same topology as the experimentally observed structures. At a fixed molecular area in the phase coexistence region, the DMPA/cholesterol system undergoes an exponential decay of the line tension λ with decreasing subphase temperature T.

Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA

PubMed Central

Kandiah, Eaazhisai; Carriel, Diego; Perard, Julien; Malet, Hélène; Bacia, Maria; Liu, Kaiyin; Chan, Sze W. S.; Houry, Walid A.; Ollagnier de Choudens, Sandrine; Elsen, Sylvie; Gutsche, Irina

2016-01-01

The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. PMID:27080013

Molecular dynamic simulation of mGluR5 amino terminal domain: essential dynamics analysis captures the agonist or antagonist behaviour of ligands.

PubMed

Casoni, Alessandro; Clerici, Francesca; Contini, Alessandro

2013-04-01

We describe the application of molecular dynamics followed by principal component analysis to study the inter-domain movements of the ligand binding domain (LBD) of mGluR5 in response to the binding of selected agonists or antagonists. Our results suggest that the method is an attractive alternative to current approaches to predict the agonist-induced or antagonist-blocked LBD responses. The ratio between the eigenvalues of the first and second eigenvectors (R1,2) is also proposed as a numerical descriptor for discriminating the ligand behavior as a mGluR5 agonist or antagonist. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of Single Nucleotide Polymorphisms on Human N-Acetyltransferase 2 Structure and Dynamics by Molecular Dynamics Simulation

PubMed Central

Rajasekaran, M.; Abirami, Santhanam; Chen, Chinpan

2011-01-01

Background Arylamine N-acetyltransferase 2 (NAT2) is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear. Methodology/Principal Findings We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential. Conclusions/Significance Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms. PMID:21980537

The relationship among multiple patient-reported outcomes measures for patients with ulcerative colitis receiving treatment with MMX ® formulated delayed-release mesalamine.

PubMed

Yarlas, Aaron; Yen, Linnette; Hodgkins, Paul

2015-03-01

Ulcerative colitis (UC) is associated with impaired health-related quality of life (HRQL) and work-related outcomes (WRO). This analysis examined correspondences among measures of HRQL and WRO in patients with UC, as well as the magnitude of each measure's responsiveness to disease activity and treatment. An open-label, prospective trial of delayed-release mesalamine tablets formulated with MMX(®) technology included 8 weeks of treatment for patients with active mild-to-moderate UC (n = 137) and 12 months of maintenance treatment for patients with quiescent UC (n = 206). Spearman correlations (ρ) measured inter-domain associations across measures of generic HRQL [12-item Short-Form Health Survey (SF-12v2)], disease-specific HRQL [Short Inflammatory Bowel Disease Questionnaire (SIBDQ)], and disease-specific WRO [Work Productivity and Activity Impairment for Specific Health Problems (WPAI:SHP)]. Responsiveness to disease activity and treatment was assessed for each instrument. Changes in scores from baseline to week 8 were moderately correlated across all instrument domains: 65 of 80 (81 %) between-instrument inter-domain correlations were of moderate magnitude (0.30 < ρ < 0.70), with an average magnitude of 0.42 [95 % confidence interval (CI) 0.38-0.46]. Associations between symptom measures were stronger for SIBDQ (|average ρ| = 0.41; 95 % CI 0.34-0.48) and WPAI:SHP (0.40; 0.30-0.47) than SF-12v2 (0.30; 0.27-0.34). SIBDQ was most sensitive to treatment [effect size (d z ) for change from baseline to week 8 = 0.62; 95 % CI 0.35-0.89], followed by WPAI:SHP (d z = 0.43; 0.32-0.54) and SF-12v2 (d z = 0.33; 0.27-0.39). While the SIBDQ showed the greatest overall responsiveness to disease activity and treatment, all three patient-reported outcomes instruments provided complementary interpretive information regarding the impact of UC treatment.

Synchronous Motions Across the Instrumental Climate Record

NASA Astrophysics Data System (ADS)

Carl, Peter

The Earth's climate system bears a rich variety of feedback mechanisms that may give rise to complex, evolving modal structures under internal and external control. Various types of synchronization may be identified in the system's motion when looking at representative time series of the instrumental period through the glasses of an advanced technique of sparse data approximation, the Matching Pursuit (MP) approach. To disentangle the emerging network of oscillatory modes to the degree that climate dynamics turns out to be separable, a large dictionary of "Gaussian logons," i.e. frequency modulated (FM) Gabor atoms, is applied. Though the extracted modes make up linear decompositions, this flexible analyzing signal matches highly nonlinear waveforms. Univariate analyses over the period 1870-1997 are presented of a set of customary time series in annual resolution, comprising global and regional climate, central European synoptic systems, German precipitation, and runoff of the Elbe river near Dresden. All the evidence from this first-generation MP-FM study, obtained in subsequent multivariate syntheses, points to dynamically excited regimes of an organized yet complex climate system under permanent change—perhaps a (pre)chaotic one at centennial timescales, suggesting a "chaos control" perspective on global climate dynamics and change. Findings and conclusions include, among others, internal structure of reconstructed insolation, the episodic nature of global warming as reflected in multidecadal temperature modes, their swarm of "interdomain" companions across the whole system that unveils an unknown regime character of interannual climate dynamics, and the apparent onset early in the 1990s of the present thermal stagnation.

Antibody proteolysis: a common picture emerging from plants

PubMed Central

Donini, Marcello; Lombardi, Raffaele; Lonoce, Chiara; Di Carli, Mariasole; Marusic, Carla; Morea, Veronica; Di Micco, Patrizio

2015-01-01

We have recently characterized the degradation profiles of 2 human IgG1 monoclonal antibodies, the tumor-targeting mAb H10 and the anti-HIV mAb 2G12. Both mAbs were produced in plants either as stable transgenics or using a transient expression system based on leaf agroinfiltration. The purified antibodies were separated by 1DE and protein bands were characterized by N-terminal sequencing. The proteolytic cleavage sites identified in the heavy chain (HC) of both antibodies were localized in 3 inter-domain regions, suggesting that the number of proteolytic cleavage events taking place in plants is limited. One of the cleavage sites, close to the hinge region, was common to both antibodies. PMID:26186119

Inflammatory Caspases: Activation and Cleavage of Gasdermin-D In Vitro and During Pyroptosis.

PubMed

Zhao, Yue; Shi, Jianjin; Shao, Feng

2018-01-01

Gasdermin-D (also known as GSDMD), the newly identified executioner of pyroptotic cell death, is cleaved by activated caspase-1 downstream of canonical inflammasome activation or caspase-4, 5, and 11 upon their ligation and activation by cytosolic LPS. Upon a single cleavage between the two domains in Gasdermin-D, the N-terminal domain binds to membrane lipids and lyses cells by forming pores of an inner diameter of 10-14 nm within the membrane. The inter-domain cleavage of Gasdermin-D is a reliable marker for the activation of inflammatory caspases and cell pyroptosis. Here, we describe the methods for examining Gasdermin-D cleavage by activated inflammatory caspases in vitro and upon inflammasome activation in vivo.

Mutation of the Salt Bridge-forming Residues in the ETV6-SAM Domain Interface Blocks ETV6-NTRK3-induced Cellular Transformation*

PubMed Central

Cetinbas, Naniye; Huang-Hobbs, Helen; Tognon, Cristina; Leprivier, Gabriel; An, Jianghong; McKinney, Steven; Bowden, Mary; Chow, Connie; Gleave, Martin; McIntosh, Lawrence P.; Sorensen, Poul H.

2013-01-01

The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99–Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation. PMID:23798677

Mutation of the salt bridge-forming residues in the ETV6-SAM domain interface blocks ETV6-NTRK3-induced cellular transformation.

PubMed

Cetinbas, Naniye; Huang-Hobbs, Helen; Tognon, Cristina; Leprivier, Gabriel; An, Jianghong; McKinney, Steven; Bowden, Mary; Chow, Connie; Gleave, Martin; McIntosh, Lawrence P; Sorensen, Poul H

2013-09-27

The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99-Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation.

Anomalous toluene transport in model segmented polyurethane-urea/clay nanocomposites.

PubMed

Rath, Sangram K; Bahadur, Jitendra; Panda, Himanshu S; Sen, Debasis; Patro, T Umasankar; S, Praveen; Patri, Manornajan; Khakhar, Devang V

2018-05-16

The kinetics of liquid solvent sorption in polymeric systems and their nanocomposites often deviate from normal Fickian behaviour. This needs to be understood and interpreted, in terms of their underlying mechanistic origins. In the present study, the results of time dependent toluene sorption measurements in model segmented polyurethane-urea/clay nanocomposites have been analysed at room temperature. The studies revealed pronounced S-shaped sorption curves and unusually higher swelling of the nanocomposites compared to the neat polyurethane-urea matrix. Dynamic mechanical analysis (DMA) and small angle X-ray scattering (SAXS) measurements on the nanocomposites in the dry and liquid toluene saturated state have been carried out. The DMA studies revealed a significant decrease in the α relaxation temperature and storage modulus of the nanocomposites in the swollen state compared to the dry samples. The SAXS results showed that the nanoclay dispersion morphology transformed from intercalation in the dry state to exfoliation in the swollen state and the interdomain distance between hard segments increased upon swelling. Thermodynamic analysis of the Flory-Huggins interaction parameter (χ) of nanocomposite/toluene systems revealed increasingly negative χ values with increased clay loading. These results imply a significant plasticization effect of toluene on the nanocomposites. An interpretation of these data, which relates the abovementioned results, is presented in the framework of differential swelling stress (DSS) induced deviation from Fickian transport characteristics. We expect that these findings and methods may provide new insight into the analysis of the solvent diffusion process in heterogeneous polymers and their nanocomposites.

Tryptophan fluorescence in the Bacillus subtilis phototropin-related protein YtvA as a marker of interdomain interaction.

PubMed

Losi, Aba; Ternelli, Elena; Gärtner, Wolfgang

2004-01-01

The Bacillus subtilis protein YtvA, related to plant phototropins (phot), binds flavin mononucleotide (FMN) within the N-terminal light, oxygen and voltage (LOV) domain. The blue light-triggered photocycle of YtvA and phot involves the reversible formation of a covalent photoadduct between FMN and a cysteine (cys) residue. YtvA contains a single tryptophan, W103, localized on the LOV domain and conserved in all phot-LOV domains. In this study, we show that the fluorescence parameters of W103 in YtvA-LOV are markedly different from those observed in the full-length YtvA. The fluorescence quantum yields are ca 0.03 and 0.08, respectively. In YtvA-LOV, the maximum is redshifted (ca 345 vs 335 nm) and the average fluorescence lifetime shorter (2.7 vs 4.7 ns). These data indicate that W103 is located in a site of tight contact between the two domains of YtvA. In the FMN-cys adduct, selective excitation of W103 at 295 nm results in minimal changes of the fluorescence parameters with respect to the dark state. On 280 nm excitation, however, there is a detectable decrease in the fluorescence emitted from tyrosines, with concomitant increase in W103 fluorescence. This effect is reversible in the dark and might arise from a light-regulated energy transfer process from a yet unidentified tyrosine to W103.

Ligand Binding to WW Tandem Domains of YAP2 Transcriptional Regulator Is Under Negative Cooperativity

PubMed Central

Schuchardt, Brett J.; Mikles, David C.; Hoang, Lawrence M.; Bhat, Vikas; McDonald, Caleb B.; Sudol, Marius; Farooq, Amjad

2014-01-01

YAP2 transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well-documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules. PMID:25283809

Mechanism of the CO-sensing heme protein CooA: new insights from the truncated heme domain and UVRR spectroscopy

PubMed Central

Ibrahim, Mohammed; Kuchinskas, Michael; Youn, Hwan; Kerby, Robert L.; Roberts, Gary P.; Poulos, Thomas L.; Spiro, Thomas G.

2007-01-01

The bacterial CO-sensing heme protein CooA activates expression of genes whose products perform CO-metabolism by binding its target DNA in response to CO binding. The required conformational change has been proposed to result from CO-induced displacement of the heme and of the adjacent C-helix, which connects the sensory and DNA-binding domains. Support for this proposal comes from UV Resonance Raman (UVRR) spectroscopy, which reveals a more hydrophobic environment for the C-helix residue Trp110 when CO binds. In addition, we find a tyrosine UVRR response, which is attributable to weakening of a Tyr55-Glu83 H-bond that anchors the proximal side of the heme. Both Trp and Tyr responses are augmented in the heme domain when the DNA-binding domain has been removed, apparently reflecting loss of the inter-domain restraint. This augmentation is abolished by a Glu83Gln substitution, which weakens the anchoring H-bond. The CO recombination rate following photolysis of the CO adduct is similar for truncated and full-length protein, though truncation does increase the rate of CO association in the absence of photolysis; together these data indicate that truncation causes a faster dissociation of the endogenous Pro2 ligand. These findings are discussed in the light of structural evidence that the N-terminal tail, once released from the heme, selects the proper orientation of the DNA-binding domain, via docking interactions. PMID:17720248

The crystal structure of the streptococcal collagen-like protein 2 globular domain from invasive M3-type group A Streptococcus shows significant similarity to immunomodulatory HIV protein gp41.

PubMed

Squeglia, Flavia; Bachert, Beth; De Simone, Alfonso; Lukomski, Slawomir; Berisio, Rita

2014-02-21

The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.

Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

PubMed

He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

2000-08-01

Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

Free energy landscapes for initiation and branching of protein aggregation.

PubMed

Zheng, Weihua; Schafer, Nicholas P; Wolynes, Peter G

2013-12-17

Experiments on artificial multidomain protein constructs have probed the early stages of aggregation processes, but structural details of the species that initiate aggregation remain elusive. Using the associative-memory, water-mediated, structure and energy model known as AWSEM, a transferable coarse-grained protein model, we performed simulations of fused constructs composed of up to four copies of the Titin I27 domain or its mutant I27* (I59E). Free energy calculations enable us to quantify the conditions under which such multidomain constructs will spontaneously misfold. Consistent with experimental results, the dimer of I27 is found to be the smallest spontaneously misfolding construct. Our results show how structurally distinct misfolded states can be stabilized under different thermodynamic conditions, and this result provides a plausible link between the single-molecule misfolding experiments under native conditions and aggregation experiments under denaturing conditions. The conditions for spontaneous misfolding are determined by the interplay among temperature, effective local protein concentration, and the strength of the interdomain interactions. Above the folding temperature, fusing additional domains to the monomer destabilizes the native state, and the entropically stabilized amyloid-like state is favored. Because it is primarily energetically stabilized, the domain-swapped state is more likely to be important under native conditions. Both protofibril-like and branching structures are found in annealing simulations starting from extended structures, and these structures suggest a possible connection between the existence of multiple amyloidogenic segments in each domain and the formation of branched, amorphous aggregates as opposed to linear fibrillar structures.

Temperature-Dependent Conformational Properties of Human Neuronal Calcium Sensor-1 Protein Revealed by All-Atom Simulations.

PubMed

Zhu, Yuzhen; Ma, Buyong; Qi, Ruxi; Nussinov, Ruth; Zhang, Qingwen

2016-04-14

Neuronal calcium sensor-1 (NCS-1) protein has orthologues from Saccharomyces cerevisiae to human with highly conserved amino acid sequences. NCS-1 is an important factor controlling the animal's response to temperature change. This leads us to investigate the temperature effects on the conformational dynamics of human NCS-1 at 310 and 316 K by all-atom molecular dynamics (MD) simulations and dynamic community network analysis. Four independent 500 ns MD simulations show that secondary structure content at 316 K is similar to that at 310 K, whereas the global protein structure is expanded. Loop 3 (L3) adopts an extended state occuping the hydrophobic crevice, and the number of suboptimal communication paths between residue D176 and V190 is reduced at 316 K. The dynamic community network analysis suggests that the interdomain correlation is weakened, and the intradomain coupling is strengthened at 316 K. The elevated temperature reduces the number of the salt bridges, especially in C-domain. This study suggests that the elevated temperature affects the conformational dynamics of human NCS-1 protein. Comparison of the structural dynamics of R102Q mutant and Δ176-190 truncated NCS-1 suggests that the structural and dynamical response of NCS-1 protein to elevated temperature may be one of its intrinsic functional properties.

Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain.

PubMed

Yao, Hongwei; Lee, Myungwoon; Liao, Shu-Yu; Hong, Mei

2016-12-13

The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no 13 C- 13 C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP-TMD assembly may facilitate the transition of the membrane from hemifusion intermediates to the fusion pore.

Crystallization, structure and dynamics of the proton-translocating P-type ATPase.

PubMed

Scarborough, G A

2000-01-01

Large single three-dimensional crystals of the dodecylmaltoside complex of the Neurospora crassa plasma membrane H(+)-ATPase (H(+) P-ATPase) can be grown in polyethylene-glycol-containing solutions optimized for moderate supersaturation of both the protein surfaces and detergent micellar region. Large two-dimensional H(+) P-ATPase crystals also grow on the surface of such mixtures and on carbon films located at such surfaces. Electron crystallographic analysis of the two-dimensional crystals grown on carbon films has recently elucidated the structure of the H(+) P-ATPase at a resolution of 0.8 nm in the membrane plane. The two-dimensional crystals comprise two offset layers of ring-shaped ATPase hexamers with their exocytoplasmic surfaces face to face. Side-to-side interactions between the cytoplasmic regions of the hexamers in each layer can be seen, and an interaction between identical exocytoplasmic loops in opposing hexamer layers holds the two layers together. Detergent rings around the membrane-embedded region of the hexamers are clearly visible, and detergent-detergent interactions between the rings are also apparent. The crystal packing forces thus comprise both protein-protein and detergent-detergent interactions, supporting the validity of the original crystallization strategy. Ten transmembrane helices in each ATPase monomer are well-defined in the structure map. They are all relatively straight, closely packed, moderately tilted at various angles with respect to a plane normal to the membrane surface and average approximately 3.5 nm in length. The transmembrane helix region is connected in at least three places to the larger cytoplasmic region, which comprises several discrete domains separated by relatively wide, deep clefts. Previous work has shown that the H(+) P-ATPase undergoes substantial conformational changes during its catalytic cycle that are not changes in secondary structure. Importantly, the results of hydrogen/deuterium exchange experiments indicate that these conformational changes are probably rigid-body interdomain movements that lead to cleft closure. When interpreted within the framework of established principles of enzyme catalysis, this information on the structure and dynamics of the H(+) P-ATPase molecule provides the basis of a rational model for the sequence of events that occurs as the ATPase proceeds through its transport cycle. The forces that drive the sequence can also be clearly stipulated. However, an understanding of the molecular mechanism of ion transport catalyzed by the H(+) P-ATPase awaits an atomic resolution structure.

Catecholaminergic polymorphic ventricular tachycardia is caused by mutation-linked defective conformational regulation of the ryanodine receptor

PubMed Central

Uchinoumi, Hitoshi; Yano, Masafumi; Suetomi, Takeshi; Ono, Makoto; Xu, Xiaojuan; Tateishi, Hiroki; Oda, Tetsuro; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Yamamoto, Takeshi; Ikeda, Yasuhiro; Ohkusa, Tomoko; Ikemoto, Noriaki; Matsuzaki, Masunori

2010-01-01

Rationale Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in a well-defined region of the cardiac type-2 ryanodine receptor (RyR2). However, the underlying mechanism by which a single mutation in such a large molecule produces drastic effects on channel function remains unresolved. Objective Using a knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we investigated the molecular mechanism by which CPVT is induced by a single point mutation within the RyR2. Methods and Results The R2474S/+ KI mice showed no apparent structural or histological abnormalities in the heart, but they showed clear indications of other abnormalities. Bidirectional or polymorphic VT was induced after exercise on a treadmill. The interaction between the N-terminal (aa 1–600) and central (aa 2000–2500) domains of the RyR2 (an intrinsic mechanism to close Ca2+ channels) was weakened (domain unzipping). Upon protein kinase A (PKA)-mediated phosphorylation of the RyR2, this domain unzipping further increased, resulting in a significant increase in the frequency of spontaneous Ca2+ transients. cAMP-induced aberrant Ca2+ release events (Ca2+ sparks/waves) occurred at much lower sarcoplasmic reticulum (SR) Ca2+ content as compared to the wild-type (WT). Addition of a domain-unzipping peptide, DPc10 (aa 2460–2495), to the WT reproduced the aforementioned abnormalities that are characteristic of the R2474S/+ KI mice. Addition of DPc10 to the (cAMP-treated) KI cardiomyocytes produced no further effect. Conclusions A single point mutation within the RyR2 sensitizes the channel to agonists and reduces the threshold of luminal [Ca2+] for activation, primarily mediated by defective inter-domain interaction within the RyR2. PMID:20224043

Evolution of Substrate Specificity within a Diverse Family of [beta/alpha]-Barrel-fold Basic Amino Acid Decarboxylases X-ray Structure Determination of Enzymes with Specificity for L-Arginine and Carboxynorspermidine

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

Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J.

2010-08-26

Pyridoxal 5{prime}-phosphate (PLP)-dependent basic amino acid decarboxylases from the {beta}/{alpha}-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-{angstrom} resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the {beta}/{alpha}-barrel domain of one subunit and the {beta}-barrel of the other. ADC contains bothmore » a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the {beta}-barrel domain we refer to as the 'specificity helix.' Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu{sup 314}, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5{prime}-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.« less

Loss of conformational stability in calmodulin upon methionine oxidation.

PubMed Central

Gao, J; Yin, D H; Yao, Y; Sun, H; Qin, Z; Schöneich, C; Williams, T D; Squier, T C

1998-01-01

We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM. PMID:9512014

New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

PubMed Central

De la Vega-Ruíz, Gustavo; Domínguez-Ramírez, Lenin; Riveros-Rosas, Héctor; Guerrero-Mendiola, Carlos; Torres-Larios, Alfredo; Hernández-Alcántara, Gloria; García-Trejo, José J.; Ramírez-Silva, Leticia

2015-01-01

Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge-independent catalysis. PMID:25811853

Dielectric relaxations and conduction mechanisms in polyether-clay composite polymer electrolytes under high carbon dioxide pressure.

PubMed

Kitajima, Shunsuke; Bertasi, Federico; Vezzù, Keti; Negro, Enrico; Tominaga, Yoichi; Di Noto, Vito

2013-10-21

The composite material P(EO/EM)-Sa consisting of synthetic saponite (Sa) dispersed in poly[ethylene oxide-co-2-(2-methoxyethoxy)ethyl glycidyl ether] (P(EO/EM)) is studied by "in situ" measurements using broadband electrical spectroscopy (BES) under pressurized CO2 to characterize the dynamic behavior of conductivity and the dielectric relaxations of the ion host polymer matrix. It is revealed that there are three dielectric relaxation processes associated with: (I) the dipolar motions in the short oxyethylene side chains of P(EO/EM) (β); and (II) the segmental motion of the main chains comprising the polyether components (αfast, αslow). αslow is attributed to the slow α-relaxation of P(EO/EM) macromolecules, which is hindered by the strong coordination interactions with the ions. Two conduction processes are observed, σDC and σID, which are attributed, respectively, to the bulk conductivity and the interdomain conductivity. The temperature dependence of conductivity and relaxation processes reveals that αfast and αslow are strongly correlated with σDC and σID. The "in situ" BES measurements under pressurized CO2 indicate a fast decrease in σDC at the initial CO2 treatment time resulting from the decrease in the concentration of polyether-M(n+) complexes, which is driven by the CO2 permeation. The relaxation frequency (fR) of αslow at the initial CO2 treatment time increases and shows a steep rise with time with the same behavior of the αfast mode. It is demonstrated that the interactions between polyether chains of P(EO/EM) and cations in the polymer electrolyte layers embedded in Sa are probably weakened by the low permittivity of CO2 (ε = 1.08). Thus, the formation of ion pairs in the polymer electrolyte domains of P(EO/EM)-Sa occurs, with a corresponding reduction in the concentration of ion carriers.

Intracellular translocation of calmodulin and Ca{sup 2+}/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes

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

Gangopadhyay, Jaya Pal, E-mail: jaya@bbri.org; Ikemoto, Noriaki; Department of Neurology, Harvard Medical School, Boston, MA 02115

We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca{sup 2+} leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 ). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in themore » nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca{sup 2+}/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca{sup 2+} transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca{sup 2+} transients results in the appearance of trains of Ca{sup 2+} spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca{sup 2+} events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program.« less

Route Flap Damping Made Usable

NASA Astrophysics Data System (ADS)

Pelsser, Cristel; Maennel, Olaf; Mohapatra, Pradosh; Bush, Randy; Patel, Keyur

The Border Gateway Protocol (BGP), the de facto inter-domain routing protocol of the Internet, is known to be noisy. The protocol has two main mechanisms to ameliorate this, MinRouteAdvertisementInterval (MRAI), and Route Flap Damping (RFD). MRAI deals with very short bursts on the order of a few to 30 seconds. RFD deals with longer bursts, minutes to hours. Unfortunately, RFD was found to severely penalize sites for being well-connected because topological richness amplifies the number of update messages exchanged. So most operators have disabled it. Through measurement, this paper explores the avenue of absolutely minimal change to code, and shows that a few RFD algorithmic constants and limits can be trivially modified, with the result being damping a non-trivial amount of long term churn without penalizing well-behaved prefixes' normal convergence process.

Molecular dynamics simulation of the Escherichia coli NikR protein: equilibrium conformational fluctuations reveal interdomain allosteric communication pathways.

PubMed

Bradley, Michael J; Chivers, Peter T; Baker, Nathan A

2008-05-16

Escherichia coli NikR is a homotetrameric Ni(2+)- and DNA-binding protein that functions as a transcriptional repressor of the NikABCDE nickel permease. The protein is composed of two distinct domains. The N-terminal 50 amino acids of each chain forms part of the dimeric ribbon-helix-helix (RHH) domains, a well-studied DNA-binding fold. The 83-residue C-terminal nickel-binding domain forms an ACT (aspartokinase, chorismate mutase, and TyrA) fold and contains the tetrameric interface. In this study, we have utilized an equilibrium molecular dynamics simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains to gain insight into the effects of Ni(2+) on DNA-binding activity. The molecular simulation data were analyzed using two different correlation measures based on fluctuations in atomic position and noncovalent contacts together with a clustering algorithm to define groups of residues with similar correlation patterns for both types of correlation measure. Based on these analyses, we have defined a series of residue interrelationships that describe an allosteric communication pathway between the Ni(2+)- and DNA-binding sites, which are separated by 40 A. Several of the residues identified by our analyses have been previously shown experimentally to be important for NikR function. An additional subset of the identified residues structurally connects the experimentally implicated residues and may help coordinate the allosteric communication between the ACT and RHH domains.

Molecular dynamics simulation of the Escherichia coli NikR protein: Equilibrium conformational fluctuations reveal inter-domain allosteric communication pathways

PubMed Central

Bradley, Michael J.; Chivers, Peter T.; Baker, Nathan A.

2008-01-01

Summary E. coliNikR is a homotetrameric Ni2+- and DNA-binding protein that functions as a transcriptional repressor of the NikABCDE nickel permease. The protein is composed of 2 distinct domains. The N-terminal fifty amino acids of each chain forms part of the dimeric ribbon-helix-helix (RHH) domains, a well-studied DNA-binding fold. The eighty-three residue C-terminal nickel-binding domain forms an ACT-fold and contains the tetrameric interface. In this study, we have utilized an equilibrium molecular dynamics (MD) simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains to gain insight into the effects of Ni on DNA-binding activity. The molecular simulation data was analyzed using two different correlation measures based on fluctuations in atomic position and non-covalent contacts, together with a clustering algorithm to define groups of residues with similar correlation patterns for both types of correlation measure. Based on these analyses, we have defined a series of residue interrelationships that describe an allosteric communication pathway between the Ni2+ and DNA binding sites, which are separated by 40 Å. Several of the residues identified by our analyses have been previously shown experimentally to be important for NikR function. An additional subset of the identified residues structurally connects the experimentally implicated residues and may help coordinate the allosteric communication between the ACT and RHH domains. PMID:18433769

Ligand binding to WW tandem domains of YAP2 transcriptional regulator is under negative cooperativity.

PubMed

Schuchardt, Brett J; Mikles, David C; Hoang, Lawrence M; Bhat, Vikas; McDonald, Caleb B; Sudol, Marius; Farooq, Amjad

2014-12-01

YES-associated protein 2 (YAP2) transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of the WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules. © 2014 FEBS.

Solution structure and backbone dynamics of the N-terminal region of the calcium regulatory domain from soybean calcium-dependent protein kinase alpha.

PubMed

Weljie, Aalim M; Gagné, Stéphane M; Vogel, Hans J

2004-12-07

Ca(2+)-dependent protein kinases (CDPKs) are vital Ca(2+)-signaling proteins in plants and protists which have both a kinase domain and a self-contained calcium regulatory calmodulin-like domain (CLD). Despite being very similar to CaM (>40% identity) and sharing the same fold, recent biochemical and structural evidence suggests that the behavior of CLD is distinct from its namesake, calmodulin. In this study, NMR spectroscopy is employed to examine the structure and backbone dynamics of a 168 amino acid Ca(2+)-saturated construct of the CLD (NtH-CLD) in which almost the entire C-terminal domain is exchange broadened and not visible in the NMR spectra. Structural characterization of the N-terminal domain indicates that the first Ca(2+)-binding loop is significantly more open than in a recently reported structure of the CLD complexed with a putative intramolecular binding region (JD) in the CDPK. Backbone dynamics suggest that parts of the third helix exhibit unusually high mobility, and significant exchange, consistent with previous findings that this helix interacts with the C-terminal domain. Dynamics data also show that the "tether" region, consisting of the first 11 amino acids of CLD, is highly mobile and these residues exhibit distinctive beta-type secondary structure, which may help to position the JD and CLD. Finally, the unusual global dynamic behavior of the protein is rationalized on the basis of possible interdomain rearrangements and the highly variable environments of the C- and N-terminal domains.

Role of κ→λ light-chain constant-domain switch in the structure and functionality of A17 reactibody

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

Ponomarenko, Natalia; Chatziefthimiou, Spyros D.; Kurkova, Inna

2014-03-01

Catalytic antibody variants with κ and λ light-chain constant domains show differences in their crystal structures which lead to subtle changes in catalytic efficiency and thermodynamic parameters as well as in their affinity for peptide substrates. The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was foundmore » that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the V{sub L} and V{sub H} domains. These V{sub L}/V{sub H} domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.« less

Refined structure of dimeric diphtheria toxin at 2.0 A resolution.

PubMed Central

Bennett, M. J.; Choe, S.; Eisenberg, D.

1994-01-01

The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed. PMID:7833807

Dynamic Motion and Communication in the Streptococcal C1 Phage Lysin, PlyC

PubMed Central

Reboul, Cyril F.; Cowieson, Nathan P.; Costa, Mauricio G. S.; Kass, Itamar; Jackson, Colin; Perahia, David; Buckle, Ashley M.; McGowan, Sheena

2015-01-01

The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria and therefore lysins represent novel antibacterial agents. The PlyC phage lysin is the most potent lysin characterized to date and can rapidly lyse Group A, C and E streptococci. Previously, we have determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the crystal structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Here we use small angle X-ray scattering (SAXS) and reveal that the conformational ensemble of PlyC in solution is different to that in the crystal structure. We also investigated the flexibility of the enzyme using both normal mode (NM) analysis and molecular dynamics (MD) simulations. Consistent with our SAXS data, MD simulations show rotational dynamics of both catalytic domains, and implicate inter-domain communication in achieving a substrate-ready conformation required for enzyme function. Our studies therefore provide insights into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency. PMID:26470022

Independent saturation of three TrpRS subsites generates a partially assembled state similar to those observed in molecular simulations

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

Laowanapiban, Poramaet; Kapustina, Maryna; Vonrhein, Clemens

2009-03-05

Two new crystal structures of Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) afford evidence that a closed interdomain hinge angle requires a covalent bond between AMP and an occupant of either pyrophosphate or tryptophan subsite. They also are within experimental error of a cluster of structures observed in a nonequilibrium molecular dynamics simulation showing partial active-site assembly. Further, the highest energy structure in a minimum action pathway computed by using elastic network models for Open and Pretransition state (PreTS) conformations for the fully liganded TrpRS monomer is intermediate between that simulated structure and a partially disassembled structure from a nonequilibrium molecular dynamicsmore » trajectory for the unliganded PreTS. These mutual consistencies provide unexpected validation of inferences drawn from molecular simulations.« less

Crystal structure of tandem type III fibronectin domains from Drosophila neuroglian at 2.0 A.

PubMed

Huber, A H; Wang, Y M; Bieber, A J; Bjorkman, P J

1994-04-01

We report the crystal structure of two adjacent fibronectin type III repeats from the Drosophila neural cell adhesion molecule neuroglian. Each domain consists of two antiparallel beta sheets and is folded topologically identically to single fibronectin type III domains from the extracellular matrix proteins tenascin and fibronectin. beta bulges and left-handed polyproline II helices disrupt the regular beta sheet structure of both neuroglian domains. The hydrophobic interdomain interface includes a metal-binding site, presumably involved in stabilizing the relative orientation between domains and predicted by sequence comparision to be present in the vertebrate homolog molecule L1. The neuroglian domains are related by a near perfect 2-fold screw axis along the longest molecular dimension. Using this relationship, a model for arrays of tandem fibronectin type III repeats in neuroglian and other molecules is proposed.

How discordant morphological and molecular evolution among microorganisms can revise our notions of biodiversity on earth

PubMed Central

Lahr, Daniel J. G.; Laughinghouse, H. Dail; Oliverio, Angela; Gao, Feng; Katz, Laura A.

2014-01-01

Microscopy has revealed a tremendous diversity of bacterial and eukaryotic forms. More recent molecular analyses show discordance in estimates of biodiversity based on morphological analyses. Moreover, phylogenetic analyses of the diversity of microbial forms have revealed evidence of convergence at scales as large as interdomain – i.e. convergent forms shared between bacteria and eukaryotes. Here, we highlight examples of such discordance, focusing on exemplary lineages such as testate amoebae, ciliates and cyanobacteria, which have long histories of morphological study. We discuss examples in two categories: 1) morphologically identical (or highly similar) individuals that are genetically distinct and 2) morphologically distinct individuals that are genetically distinct. We argue that hypotheses about discordance can be tested using the concept of neutral morphologies, or more broadly neutral phenotypes, as a null hypothesis. PMID:25156897

Peering Strategic Game Models for Interdependent ISPs in Content Centric Internet

PubMed Central

Guan, Jianfeng; Xu, Changqiao; Su, Wei; Zhang, Hongke

2013-01-01

Emergent content-oriented networks prompt Internet service providers (ISPs) to evolve and take major responsibility for content delivery. Numerous content items and varying content popularities motivate interdependence between peering ISPs to elaborate their content caching and sharing strategies. In this paper, we propose the concept of peering for content exchange between interdependent ISPs in content centric Internet to minimize content delivery cost by a proper peering strategy. We model four peering strategic games to formulate four types of peering relationships between ISPs who are characterized by varying degrees of cooperative willingness from egoism to altruism and interconnected as profit-individuals or profit-coalition. Simulation results show the price of anarchy (PoA) and communication cost in the four games to validate that ISPs should decide their peering strategies by balancing intradomain content demand and interdomain peering relations for an optimal cost of content delivery. PMID:24381517

Amyloid Fiber Formation in Human γD-Crystallin Induced by UV-B Photodamage

PubMed Central

Moran, Sean D.; Zhang, Tianqi O.; Decatur, Sean M.; Zanni, Martin T.

2013-01-01

γD-crystallin is an abundant structural protein of the lens that is found in native and modified forms in cataractous aggregates. We establish that UV-B irradiation of γD-crystallin leads to structurally specific modifications and precipitation via two mechanisms: amorphous aggregates and amyloid fibers. UV-B radiation causes cleavage of the backbone, in large measure near the interdomain interface, where side chain oxidations are also concentrated. 2D IR spectroscopy and expressed protein ligation localize fiber formation exclusively to the C-terminal domain of γD-crystallin. The native β-sandwich domains are not retained upon precipitation by either mechanism. The similarity between the amyloid forming pathway when induced by either UV-B radiation or low pH suggests that it is the propensity for the C-terminal β-sandwich domain to form amyloid β-sheets that determines the misfolding pathway independent of the mechanism of denaturation. PMID:23957864

Transformation of BCC and B2 High Temperature Phases to HCP and Orthorhombic Structures in the Ti-Al-Nb System. Part I: Microstructural Predictions Based on a Subgroup Relation Between Phases

PubMed Central

Bendersky, L. A.; Roytburd, A.; Boettinger, W. J.

1993-01-01

Possible paths for the constant composition coherent transformation of BCC or B2 high temperature phases to low temperature HCP or Orthorhombic phases in the Ti-Al-Nb system are analyzed using a sequence of ciystallographic structural relationships developed from subgroup symmetry relations. Symmetry elements lost in each step of the sequence determine the possibilities for variants of the low symmetry phase and domains that can be present in the microstructure. The orientation of interdomain interfaces is determined by requiring the existence of a strain-free interface between the domains. Polydomain structures are also determined that minimize elastic energy. Microstructural predictions are made for comparison to experimental results given by Benderslcy and Boettinger [J. Res. Natl. Inst. Stand. Technol. 98, 585 (1993)]. PMID:28053487

Peering strategic game models for interdependent ISPs in content centric Internet.

PubMed

Zhao, Jia; Guan, Jianfeng; Xu, Changqiao; Su, Wei; Zhang, Hongke

2013-01-01

Emergent content-oriented networks prompt Internet service providers (ISPs) to evolve and take major responsibility for content delivery. Numerous content items and varying content popularities motivate interdependence between peering ISPs to elaborate their content caching and sharing strategies. In this paper, we propose the concept of peering for content exchange between interdependent ISPs in content centric Internet to minimize content delivery cost by a proper peering strategy. We model four peering strategic games to formulate four types of peering relationships between ISPs who are characterized by varying degrees of cooperative willingness from egoism to altruism and interconnected as profit-individuals or profit-coalition. Simulation results show the price of anarchy (PoA) and communication cost in the four games to validate that ISPs should decide their peering strategies by balancing intradomain content demand and interdomain peering relations for an optimal cost of content delivery.

Fast-responding bio-based shape memory thermoplastic polyurethanes

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

Petrovic, Zoran S.; Milic, Jelena; Zhang, Fan

Fast response shape-memory polyurethanes were prepared from bio-based polyols, diphenyl methane diisocyanate and butane diol. The bio-based polyester polyols were synthesized from 9-hydroxynonanoic acid, a product obtained by ozonolysis of fatty acids extracted from soy oil and castor oil. The morphology of polyurethanes was investigated by synchrotron ultra-small angle X-ray scattering, which revealed the inter-domain spacing between the hard and soft phases, the degree of phase separation, and the level of intermixing between the hard and soft phases. We also conducted thorough investigations of the thermal, mechanical, and dielectric properties of the polyurethanes, and found that high crystallization rate ofmore » the soft segment gives these polyurethanes unique properties suitable for shapememory applications, such as adjustable transition temperatures, high degree of elastic elongations, and good mechanical strength. In conclusion, these materials are also potentially biodegradable and biocompatible, therefore suitable for biomedical and environmental applications.« less

Psychometric Properties of the Self-Perception Profile for Children in Children with Chronic Illness.

PubMed

Ferro, Mark A; Tang, Jennie

2017-07-01

The Self-Perception Profile for Children (SPPC) is a commonly used measure of self-concept in children, but little research has examined its psychometric properties in children newly-diagnosed with chronic illness. Confirmatory factor analysis and examination of reliability and convergent and discriminant validity of the SPPC was conducted in 31 children newly-diagnosed with asthma, diabetes, epilepsy, food allergy, or juvenile arthritis. The unidimensionality of each domain of the SPPC was confirmed, internal reliability was robust (α=.83-.95), and inter-domain polychoric correlations ranged from weak to strong (ρ=.05-.85) Convergent validity was demonstrated with measures of global self-concept and domains of quality of life. The Global Self-worth domain showed discriminant validity between children with and without comorbid mental disorder. Findings extend the psychometric properties of the SPPC as a valid and reliable scale in children newly-diagnosed with chronic illness.

Fast-responding bio-based shape memory thermoplastic polyurethanes

DOE PAGES

Petrovic, Zoran S.; Milic, Jelena; Zhang, Fan; ...

2017-05-31

Fast response shape-memory polyurethanes were prepared from bio-based polyols, diphenyl methane diisocyanate and butane diol. The bio-based polyester polyols were synthesized from 9-hydroxynonanoic acid, a product obtained by ozonolysis of fatty acids extracted from soy oil and castor oil. The morphology of polyurethanes was investigated by synchrotron ultra-small angle X-ray scattering, which revealed the inter-domain spacing between the hard and soft phases, the degree of phase separation, and the level of intermixing between the hard and soft phases. We also conducted thorough investigations of the thermal, mechanical, and dielectric properties of the polyurethanes, and found that high crystallization rate ofmore » the soft segment gives these polyurethanes unique properties suitable for shapememory applications, such as adjustable transition temperatures, high degree of elastic elongations, and good mechanical strength. In conclusion, these materials are also potentially biodegradable and biocompatible, therefore suitable for biomedical and environmental applications.« less

The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

NASA Astrophysics Data System (ADS)

Hu, Xiaohu; Hong, Liang; Dean Smith, Micholas; Neusius, Thomas; Cheng, Xiaolin; Smith, Jeremy C.

2016-02-01

Internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behaviour with effective relaxation times existing over many decades in time, from ps up to ~102 s (refs ,,,). Here, using molecular dynamics simulations, we show that, on timescales from 10-12 to 10-5 s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of the energy landscape explored. Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behaviour persists up to timescales approaching the in vivo lifespan of individual protein molecules.

Fast-Responding Bio-Based Shape Memory Thermoplastic Polyurethanes.

PubMed

Petrović, Zoran S; Milić, Jelena; Zhang, Fan; Ilavsky, Jan

2017-07-14

Novel fast response shape-memory polyurethanes were prepared from bio-based polyols, diphenyl methane diisocyanate and butane diol for the first time. The bio-based polyester polyols were synthesized from 9-hydroxynonanoic acid, a product obtained by ozonolysis of fatty acids extracted from soy oil and castor oil. The morphology of polyurethanes was investigated by synchrotron ultra-small angle X-ray scattering, which revealed the inter-domain spacing between the hard and soft phases, the degree of phase separation, and the level of intermixing between the hard and soft phases. We also conducted thorough investigations of the thermal, mechanical, and dielectric properties of the polyurethanes, and found that high crystallization rate of the soft segment gives these polyurethanes unique properties suitable for shape-memory applications, such as adjustable transition temperatures, high degree of elastic elongations, and good mechanical strength. These materials are also potentially biodegradable and biocompatible, therefore suitable for biomedical and environmental applications.

Identification of novel Trypanosoma cruzi prolyl oligopeptidase inhibitors by structure-based virtual screening

NASA Astrophysics Data System (ADS)

de Almeida, Hugo; Leroux, Vincent; Motta, Flávia Nader; Grellier, Philippe; Maigret, Bernard; Santana, Jaime M.; Bastos, Izabela Marques Dourado

2016-12-01

We have previously demonstrated that the secreted prolyl oligopeptidase of Trypanosoma cruzi (POPTc80) is involved in the infection process by facilitating parasite migration through the extracellular matrix. We have built a 3D structural model where POPTc80 is formed by a catalytic α/β-hydrolase domain and a β-propeller domain, and in which the substrate docks at the inter-domain interface, suggesting a "jaw opening" gating access mechanism. This preliminary model was refined by molecular dynamics simulations and next used for a virtual screening campaign, whose predictions were tested by standard binding assays. This strategy was successful as all 13 tested molecules suggested from the in silico calculations were found out to be active POPTc80 inhibitors in the micromolar range (lowest K i at 667 nM). This work paves the way for future development of innovative drugs against Chagas disease.

Continually emerging mechanistic complexity of the multi-enzyme cellulosome complex.

PubMed

Smith, Steven P; Bayer, Edward A; Czjzek, Mirjam

2017-06-01

The robust plant cell wall polysaccharide-degrading properties of anaerobic bacteria are harnessed within elegant, marcomolecular assemblages called cellulosomes, in which proteins of complementary activities amass on scaffold protein networks. Research efforts have focused and continue to focus on providing detailed mechanistic insights into cellulosomal complex assembly, topology, and function. The accumulated information is expanding our fundamental understanding of the lignocellulosic biomass decomposition process and enhancing the potential of engineered cellulosomal systems for biotechnological purposes. Ongoing biochemical studies continue to reveal unexpected functional diversity within traditional cellulase families. Genomic, proteomic, and functional analyses have uncovered unanticipated cellulosomal proteins that augment the function of the native and designer cellulosomes. In addition, complementary structural and computational methods are continuing to provide much needed insights on the influence of cellulosomal interdomain linker regions on cellulosomal assembly and activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

NASA Astrophysics Data System (ADS)

Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

2015-07-01

A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

Chemical shift imprint of intersubunit communication in a symmetric homodimer

PubMed Central

Falk, Bradley T.; Sapienza, Paul J.; Lee, Andrew L.

2016-01-01

Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein–ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state (“lig1”) of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP1 peaks are found beyond the limits set by apo and dUMP2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers. PMID:27466406

Phylogenetic and specificity studies of two-domain GNA-related lectins: generation of multispecificity through domain duplication and divergent evolution

PubMed Central

Van Damme, Els J. M.; Nakamura-Tsuruta, Sachiko; Smith, David F.; Ongenaert, Maté; Winter, Harry C.; Rougé, Pierre; Goldstein, Irwin J.; Mo, Hanqing; Kominami, Junko; Culerrier, Raphaël; Barre, Annick; Hirabayashi, Jun; Peumans, Willy J.

2007-01-01

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes. PMID:17288538

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes*

PubMed Central

Lopata, Anna; Leveles, Ibolya; Bendes, Ábris Ádám; Viskolcz, Béla; Vértessy, Beáta G.; Jójárt, Balázs; Tóth, Judit

2016-01-01

dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis. In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent. PMID:27815500

A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo.

PubMed

Ruminski, Dana J; Watson, Peter Y; Mahen, Elisabeth M; Fedor, Martha J

2016-03-01

RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo. © 2016 Ruminski et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities*

PubMed Central

Lim, Siew Pheng; Koh, Jolene Hong Kiew; Seh, Cheah Chen; Liew, Chong Wai; Davidson, Andrew D.; Chua, Leng Shiew; Chandrasekaran, Ramya; Cornvik, Tobias C.; Shi, Pei-Yong; Lescar, Julien

2013-01-01

The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263–271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1–4 proteins and improves their de novo polymerase initiation activities by enhancing the turnover of the RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication. We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265–900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272–900) reported previously. This new orthorhombic crystal form (space group P21212) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed “preinitiation” conformation similar to the one that was captured previously in space group C2221 with one molecule per asymmetric unit. The structure reveals that residues 269–271 interact with the RdRp domain and suggests that residues 263–268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp. PMID:24025331

Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

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

Kirkensgaard, Kristine G.; Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark; Hägglund, Per

2009-09-01

The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR,more » HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R{sub cryst} of 19.0% and an R{sub free} of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation.« less

The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

DOE PAGES

Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; ...

2014-08-11

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R 2:C 2), with a regulatory subunit homodimer (R 2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the typemore » IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.« less

Human Neuronal Calcium Sensor-1 Protein Avoids Histidine Residues To Decrease pH Sensitivity.

PubMed

Gong, Yehong; Zhu, Yuzhen; Zou, Yu; Ma, Buyong; Nussinov, Ruth; Zhang, Qingwen

2017-01-26

pH is highly regulated in mammalian central nervous systems. Neuronal calcium sensor-1 (NCS-1) can interact with numerous target proteins. Compared to that in the NCS-1 protein of Caenorhabditis elegans, evolution has avoided the placement of histidine residues at positions 102 and 83 in the NCS-1 protein of humans and Xenopus laevis, possibly to decrease the conformational sensitivity to pH gradients in synaptic processes. We used all-atom molecular dynamics simulations to investigate the effects of amino acid substitutions between species on human NCS-1 by substituting Arg102 and Ser83 for histidine at neutral (R102H and S83H) and acidic pHs (R102H p and S83H p ). Our cumulative 5 μs simulations revealed that the R102H mutation slightly increases the structural flexibility of loop L2 and the R102H p mutation decreases protein stability. Community network analysis illustrates that the R102H and S83H mutations weaken the interdomain and strengthen the intradomain communications. Secondary structure contents in the S83H and S83H p mutants are similar to those in the wild type, whereas the global structural stabilities and salt-bridge probabilities decrease. This study highlights the conformational dynamics effects of the R102H and S83H mutations on the local structural flexibility and global stability of NCS-1, whereas protonated histidine decreases the stability of NCS-1. Thus, histidines at positions 102 and 83 may not be compatible with the function of NCS-1 whether in the neutral or protonated state.

Independent Structural Domains in Paramyxovirus Polymerase Protein*

PubMed Central

Dochow, Melanie; Krumm, Stefanie A.; Crowe, James E.; Moore, Martin L.; Plemper, Richard K.

2012-01-01

All enzymatic activities required for genomic replication and transcription of nonsegmented negative strand RNA viruses (or Mononegavirales) are believed to be concentrated in the viral polymerase (L) protein. However, our insight into the organization of these different enzymatic activities into a bioactive tertiary structure remains rudimentary. Fragments of Mononegavirales polymerases analyzed to date cannot restore bioactivity through trans-complementation, unlike the related L proteins of segmented NSVs. We investigated the domain organization of phylogenetically diverse Paramyxovirus L proteins derived from measles virus (MeV), Nipah virus (NiV), and respiratory syncytial virus (RSV). Through a comprehensive in silico and experimental analysis of domain intersections, we defined MeV L position 615 as an interdomain candidate in addition to the previously reported residue 1708. Only position 1708 of MeV and the homologous positions in NiV and RSV L also tolerated the insertion of epitope tags. Splitting of MeV L at residue 1708 created fragments that were unable to physically interact and trans-complement, but strikingly, these activities were reconstituted by the addition of dimerization tags to the fragments. Equivalently split fragments of NiV, RSV, and MeV L oligomerized with comparable efficiency in all homo- and heterotypic combinations, but only the homotypic pairs were able to trans-complement. These results demonstrate that synthesis as a single polypeptide is not required for the Mononegavirales polymerases to adopt a proper tertiary conformation. Paramyxovirus polymerases are composed of at least two truly independent folding domains that lack a traditional interface but require molecular compatibility for bioactivity. The functional probing of the L domain architecture through trans-complementation is anticipated to be applicable to all Mononegavirales polymerases. PMID:22215662

Structural mechanisms of chaperone mediated protein disaggregation

PubMed Central

Sousa, Rui

2014-01-01

The ClpB/Hsp104 and Hsp70 classes of molecular chaperones use ATP hydrolysis to dissociate protein aggregates and complexes, and to move proteins through membranes. ClpB/Hsp104 are members of the AAA+ family of proteins which form ring-shaped hexamers. Loops lining the pore in the ring engage substrate proteins as extended polypeptides. Interdomain rotations and conformational changes in these loops coupled to ATP hydrolysis unfold and pull proteins through the pore. This provides a mechanism that progressively disrupts local secondary and tertiary structure in substrates, allowing these chaperones to dissociate stable aggregates such as β-sheet rich prions or coiled coil SNARE complexes. While the ClpB/Hsp104 mechanism appears to embody a true power-stroke in which an ATP powered conformational change in one protein is directly coupled to movement or structural change in another, the mechanism of force generation by Hsp70s is distinct and less well understood. Both active power-stroke and purely passive mechanisms in which Hsp70 captures spontaneous fluctuations in a substrate have been proposed, while a third proposed mechanism—entropic pulling—may be able to generate forces larger than seen in ATP-driven molecular motors without the conformational coupling required for a power-stroke. The disaggregase activity of these chaperones is required for thermotolerance, but unrestrained protein complex/aggregate dissociation is potentially detrimental. Disaggregating chaperones are strongly auto-repressed, and are regulated by co-chaperones which recruit them to protein substrates and activate the disaggregases via mechanisms involving either sequential transfer of substrate from one chaperone to another and/or simultaneous interaction of substrate with multiple chaperones. By effectively subjecting substrates to multiple levels of selection by multiple chaperones, this may insure that these potent disaggregases are only activated in the appropriate context. PMID:25988153

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family.

PubMed

Shaner, Lance; Trott, Amy; Goeckeler, Jennifer L; Brodsky, Jeffrey L; Morano, Kevin A

2004-05-21

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.

Atomistic modeling of alternating access of a mitochondrial ADP/ATP membrane transporter with molecular simulations

PubMed Central

Hayashi, Shigehiko

2017-01-01

The mitochondrial ADP/ATP carrier (AAC) is a membrane transporter that exchanges a cytosolic ADP for a matrix ATP. Atomic structures in an outward-facing (OF) form which binds an ADP from the intermembrane space have been solved by X-ray crystallography, and revealed their unique pseudo three-fold symmetry fold which is qualitatively different from pseudo two-fold symmetry of most transporters of which atomic structures have been solved. However, any atomic-level information on an inward-facing (IF) form, which binds an ATP from the matrix side and is fixed by binding of an inhibitor, bongkrekic acid (BA), is not available, and thus its alternating access mechanism for the transport process is unknown. Here, we report an atomic structure of the IF form predicted by atomic-level molecular dynamics (MD) simulations of the alternating access transition with a recently developed accelerating technique. We successfully obtained a significantly stable IF structure characterized by newly formed well-packed and -organized inter-domain interactions through the accelerated simulations of unprecedentedly large conformational changes of the alternating access without a prior knowledge of the target protein structure. The simulation also shed light on an atomistic mechanism of the strict transport selectivity of adenosine nucleotides over guanosine and inosine ones. Furthermore, the IF structure was shown to bind ATP and BA, and thus revealed their binding mechanisms. The present study proposes a qualitatively novel view of the alternating access of transporters having the unique three-fold symmetry in atomic details and opens the way for rational drug design targeting the transporter in the dynamic functional cycle. PMID:28727843

Combination of X-ray crystallography, SAXS and DEER to obtain the structure of the FnIII-3, 4 domains of integrin α6β4

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

Alonso-García, Noelia; García-Rubio, Inés; Academia General Militar, Carretera de Huesca s/n, 50090 Zaragoza

The structure of the FnIII-3, 4 region of integrin β4 was solved using a hybrid approach that combines crystallographic structures, SAXS, DEER and molecular modelling. The structure helps in understanding how integrin β4 might bind to other hemidesmosomal proteins and mediate signalling. Integrin α6β4 is a major component of hemidesmosomes that mediate the stable anchorage of epithelial cells to the underlying basement membrane. Integrin α6β4 has also been implicated in cell proliferation and migration and in carcinoma progression. The third and fourth fibronectin type III domains (FnIII-3, 4) of integrin β4 mediate binding to the hemidesmosomal proteins BPAG1e and BPAG2,more » and participate in signalling. Here, it is demonstrated that X-ray crystallography, small-angle X-ray scattering and double electron–electron resonance (DEER) complement each other to solve the structure of the FnIII-3, 4 region. The crystal structures of the individual FnIII-3 and FnIII-4 domains were solved and the relative arrangement of the FnIII domains was elucidated by combining DEER with site-directed spin labelling. Multiple structures of the interdomain linker were modelled by Monte Carlo methods complying with DEER constraints, and the final structures were selected against experimental scattering data. FnIII-3, 4 has a compact and cambered flat structure with an evolutionary conserved surface that is likely to correspond to a protein-interaction site. Finally, this hybrid method is of general application for the study of other macromolecules and complexes.« less

The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

PubMed

Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

2014-10-10

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium.

PubMed

Shishkov, Alexander; Bogacheva, Elena; Fedorova, Natalia; Ksenofontov, Alexander; Badun, Gennadii; Radyukhin, Victor; Lukashina, Elena; Serebryakova, Marina; Dolgov, Alexey; Chulichkov, Alexey; Dobrov, Evgeny; Baratova, Lyudmila

2011-12-01

The structure of the C-terminal domain of the influenza virus A matrix M1 protein, for which X-ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally-activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C-terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C-terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C-terminal domain is an almost flat layer with a three-α-helical structure. To explain the high level of tritium label incorporation into the C-terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI-TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C-terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell. © 2011 The Authors Journal compilation © 2011 FEBS.

Network-wide BGP route prediction for traffic engineering

NASA Astrophysics Data System (ADS)

Feamster, Nick; Rexford, Jennifer

2002-07-01

The Internet consists of about 13,000 Autonomous Systems (AS's) that exchange routing information using the Border Gateway Protocol (BGP). The operators of each AS must have control over the flow of traffic through their network and between neighboring AS's. However, BGP is a complicated, policy-based protocol that does not include any direct support for traffic engineering. In previous work, we have demonstrated that network operators can adapt the flow of traffic in an efficient and predictable fashion through careful adjustments to the BGP policies running on their edge routers. Nevertheless, many details of the BGP protocol and decision process make predicting the effects of these policy changes difficult. In this paper, we describe a tool that predicts traffic flow at network exit points based on the network topology, the import policy associated with each BGP session, and the routing advertisements received from neighboring AS's. We present a linear-time algorithm that computes a network-wide view of the best BGP routes for each destination prefix given a static snapshot of the network state, without simulating the complex details of BGP message passing. We describe how to construct this snapshot using the BGP routing tables and router configuration files available from operational routers. We verify the accuracy of our algorithm by applying our tool to routing and configuration data from AT&T's commercial IP network. Our route prediction techniques help support the operation of large IP backbone networks, where interdomain routing is an important aspect of traffic engineering.

A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity.

PubMed

Mills, Jamie E; Whitford, Paul C; Shaffer, Jennifer; Onuchic, Jose N; Adams, Joseph A; Jennings, Patricia A

2007-02-02

The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.

Database Resources of the BIG Data Center in 2018

PubMed Central

Xu, Xingjian; Hao, Lili; Zhu, Junwei; Tang, Bixia; Zhou, Qing; Song, Fuhai; Chen, Tingting; Zhang, Sisi; Dong, Lili; Lan, Li; Wang, Yanqing; Sang, Jian; Hao, Lili; Liang, Fang; Cao, Jiabao; Liu, Fang; Liu, Lin; Wang, Fan; Ma, Yingke; Xu, Xingjian; Zhang, Lijuan; Chen, Meili; Tian, Dongmei; Li, Cuiping; Dong, Lili; Du, Zhenglin; Yuan, Na; Zeng, Jingyao; Zhang, Zhewen; Wang, Jinyue; Shi, Shuo; Zhang, Yadong; Pan, Mengyu; Tang, Bixia; Zou, Dong; Song, Shuhui; Sang, Jian; Xia, Lin; Wang, Zhennan; Li, Man; Cao, Jiabao; Niu, Guangyi; Zhang, Yang; Sheng, Xin; Lu, Mingming; Wang, Qi; Xiao, Jingfa; Zou, Dong; Wang, Fan; Hao, Lili; Liang, Fang; Li, Mengwei; Sun, Shixiang; Zou, Dong; Li, Rujiao; Yu, Chunlei; Wang, Guangyu; Sang, Jian; Liu, Lin; Li, Mengwei; Li, Man; Niu, Guangyi; Cao, Jiabao; Sun, Shixiang; Xia, Lin; Yin, Hongyan; Zou, Dong; Xu, Xingjian; Ma, Lina; Chen, Huanxin; Sun, Yubin; Yu, Lei; Zhai, Shuang; Sun, Mingyuan; Zhang, Zhang; Zhao, Wenming; Xiao, Jingfa; Bao, Yiming; Song, Shuhui; Hao, Lili; Li, Rujiao; Ma, Lina; Sang, Jian; Wang, Yanqing; Tang, Bixia; Zou, Dong; Wang, Fan

2018-01-01

Abstract The BIG Data Center at Beijing Institute of Genomics (BIG) of the Chinese Academy of Sciences provides freely open access to a suite of database resources in support of worldwide research activities in both academia and industry. With the vast amounts of omics data generated at ever-greater scales and rates, the BIG Data Center is continually expanding, updating and enriching its core database resources through big-data integration and value-added curation, including BioCode (a repository archiving bioinformatics tool codes), BioProject (a biological project library), BioSample (a biological sample library), Genome Sequence Archive (GSA, a data repository for archiving raw sequence reads), Genome Warehouse (GWH, a centralized resource housing genome-scale data), Genome Variation Map (GVM, a public repository of genome variations), Gene Expression Nebulas (GEN, a database of gene expression profiles based on RNA-Seq data), Methylation Bank (MethBank, an integrated databank of DNA methylomes), and Science Wikis (a series of biological knowledge wikis for community annotations). In addition, three featured web services are provided, viz., BIG Search (search as a service; a scalable inter-domain text search engine), BIG SSO (single sign-on as a service; a user access control system to gain access to multiple independent systems with a single ID and password) and Gsub (submission as a service; a unified submission service for all relevant resources). All of these resources are publicly accessible through the home page of the BIG Data Center at http://bigd.big.ac.cn. PMID:29036542

A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production.

PubMed

Gebhard, Leopoldo G; Iglesias, Néstor G; Byk, Laura A; Filomatori, Claudia V; De Maio, Federico A; Gamarnik, Andrea V

2016-06-01

Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. Dengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for dengue virus particle assembly. NS3 is a multifunctional enzyme that participates in different steps of the viral life cycle. Using reporter systems to dissect different viral processes, we identified a novel N-terminal unstructured region of the NS3 protein as crucial for production of viral particles. Based on our findings, we propose new ideas that include NS3 as a possible scaffold for the viral assembly process. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.

PubMed

Yang, Yuting; Gourinath, S; Kovács, Mihály; Nyitray, László; Reutzel, Robbie; Himmel, Daniel M; O'Neall-Hennessey, Elizabeth; Reshetnikova, Ludmilla; Szent-Györgyi, Andrew G; Brown, Jerry H; Cohen, Carolyn

2007-05-01

Unlike processive cellular motors such as myosin V, whose structure has recently been determined in a "rigor-like" conformation, myosin II from contracting muscle filaments necessarily spends most of its time detached from actin. By using squid and sea scallop sources, however, we have now obtained similar rigor-like atomic structures for muscle myosin heads (S1). The significance of the hallmark closed actin-binding cleft in these crystal structures is supported here by actin/S1-binding studies. These structures reveal how different duty ratios, and hence cellular functions, of the myosin isoforms may be accounted for, in part, on the basis of detailed differences in interdomain contacts. Moreover, the rigor-like position of switch II turns out to be unique for myosin V. The overall arrangements of subdomains in the motor are relatively conserved in each of the known contractile states, and we explore qualitatively the energetics of these states.

The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

DOE PAGES

Hu, Xiaohu; Hong, Liang; Smith, Micholas Dean; ...

2015-11-23

Here, internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behavior with effective relaxation times existing over many decades in time, from ps up to ~10 2s (refs 1-4). Here, using molecular dynamics simulations, we show that, on timescales from 10 –12 to 10 –5s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of themore » energy landscape explored. Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behavior persists up to timescales approaching the in vivo lifespan of individual protein molecules.« less

Structural insights into the cofactor-assisted substrate recognition of yeast methylglyoxal/isovaleraldehyde reductase Gre2.

PubMed

Guo, Peng-Chao; Bao, Zhang-Zhi; Ma, Xiao-Xiao; Xia, Qingyou; Li, Wei-Fang

2014-09-01

Saccharomyces cerevisiae Gre2 (EC1.1.1.283) serves as a versatile enzyme that catalyzes the stereoselective reduction of a broad range of substrates including aliphatic and aromatic ketones, diketones, as well as aldehydes, using NADPH as the cofactor. Here we present the crystal structures of Gre2 from S. cerevisiae in an apo-form at 2.00Å and NADPH-complexed form at 2.40Å resolution. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain and a variable C-terminal domain, which participates in substrate recognition. The induced fit upon binding to the cofactor NADPH makes the two domains shift toward each other, producing an interdomain cleft that better fits the substrate. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis enabled us to define a potential substrate-binding pocket that determines the stringent substrate stereoselectivity for catalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Human heavy chain disease protein WIS: implications for the organization of immunoglobulin genes.

PubMed Central

Franklin, E C; Prelli, F; Frangione, B

1979-01-01

Protein WIS is a human gamma3 heavy (H) chain disease immunoglobulin variant whose amino acid sequence is most readily interpreted by postulating that three residues of the amino terminus are followed by a deletion of most of the variable (VH) domain, which ends at the variable-constant (VC) joining region. Then there is a stretch of eight residues, three of which are unusual, while the other five have striking homology to the VC junction sequence. This is followed by a second deletion, which ends at the beginning of the quadruplicated hinge region. These findings are consistent with mutations resulting in deletions of most of the gene coding for the V region and CH1 domain followed by splicing at the VC joining region and at the hinge. These structural features fit well the notion of genetic discontinuity between V and C genes and also suggest similar mechanisms of excision and splicing in the interdomain regions of the C gene of the heavy chain. PMID:106391

Parkin-phosphoubiquitin complex reveals a cryptic ubiquitin binding site required for RBR ligase activity

PubMed Central

Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

2017-01-01

RING-BETWEENRING-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR is Parkin, mutations in which lead to early onset hereditary Parkinsonism. Parkin and other RBRs share a catalytic RBR module, but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during activation of Parkin. However, current data on active RBRs are in the absence of regulatory domains. Therefore, how individual RBRs are activated, and whether they share a common mechanism remains unclear. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces a movement in the IBR domain to reveal a cryptic ubiquitin binding site. Mutation of this site negatively impacts on Parkin’s activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, suggesting a role for interdomain association in RBR ligase mechanism. PMID:28414322

Hsp90 shapes protein and RNA evolution to balance trade-offs between protein stability and aggregation.

PubMed

Geller, Ron; Pechmann, Sebastian; Acevedo, Ashley; Andino, Raul; Frydman, Judith

2018-05-03

Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis.

Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication

NASA Astrophysics Data System (ADS)

Rustiguel, Joane K.; Soares, Ricardo O. S.; Meisburger, Steve P.; Davis, Katherine M.; Malzbender, Kristina L.; Ando, Nozomi; Dias-Baruffi, Marcelo; Nonato, Maria Cristina

2016-09-01

Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

Structural basis for gating and activation of RyR1

PubMed Central

des Georges, Amédée; Clarke, Oliver B.; Zalk, Ran; Yuan, Qi; Condon, Kendall J.; Grassucci, Robert A.; Hendrickson, Wayne A.; Marks, Andrew R.; Frank, Joachim

2016-01-01

Summary The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel required for skeletal muscle contraction. Here we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca2+, ATP and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca2+ alone induce conformational changes in the cytoplasmic assembly (‘priming’), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement and deformation of the S4-S5 linker, and conformational changes in the pseudo-voltage-sensor domain. PMID:27662087

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding.

PubMed

Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D; Heywood, Sam; Humphreys, David P

2016-10-01

An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding

PubMed Central

Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E.; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D.; Heywood, Sam; Humphreys, David P.

2016-01-01

ABSTRACT An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG. PMID:27532598

Archaeal MCM has separable processivity, substrate choice and helicase domains

PubMed Central

Barry, Elizabeth R.; McGeoch, Adam T.; Kelman, Zvi; Bell, Stephen D.

2007-01-01

The mini-chromosome maintenance (MCM) complex is the principal candidate for the replicative helicase of archaea and eukaryotes. Here, we describe a functional dissection of the roles of the three principal structural modules of the homomultimeric MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results include the first analysis of the central AAA+ domain in isolation. This domain possesses ATPase and helicase activity, defining this as the minimal helicase domain. Reconstitution experiments show that the helicase activity of the AAA+ domain can be stimulated by addition of the isolated N-terminal half in trans. Addition of the N-terminus influences both the processivity of the helicase and the choice of substrate that can be melted by the ATPase domain. The degenerate helix-turn-helix domain at the C-terminus of MCM exerts a negative effect on the helicase activity of the complex. These results provide the first evidence for extensive regulatory inter-domain communication within the MCM complex. PMID:17259218

Method for identification of rigid domains and hinge residues in proteins based on exhaustive enumeration.

PubMed

Sim, Jaehyun; Sim, Jun; Park, Eunsung; Lee, Julian

2015-06-01

Many proteins undergo large-scale motions where relatively rigid domains move against each other. The identification of rigid domains, as well as the hinge residues important for their relative movements, is important for various applications including flexible docking simulations. In this work, we develop a method for protein rigid domain identification based on an exhaustive enumeration of maximal rigid domains, the rigid domains not fully contained within other domains. The computation is performed by mapping the problem to that of finding maximal cliques in a graph. A minimal set of rigid domains are then selected, which cover most of the protein with minimal overlap. In contrast to the results of existing methods that partition a protein into non-overlapping domains using approximate algorithms, the rigid domains obtained from exact enumeration naturally contain overlapping regions, which correspond to the hinges of the inter-domain bending motion. The performance of the algorithm is demonstrated on several proteins. © 2015 Wiley Periodicals, Inc.

Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity.

PubMed

Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

2017-05-01

RING-between-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR ligase is Parkin, mutations in which lead to early-onset hereditary Parkinsonism. Parkin and other RBR ligases share a catalytic RBR module but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during Parkin activation. However, current data on active RBR ligases reflect the absence of regulatory domains. Therefore, it remains unclear how individual RBR ligases are activated, and whether they share a common mechanism. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces movement in the 'in-between RING' (IBR) domain to reveal a cryptic ubiquitin-binding site. Mutation of this site negatively affects Parkin's activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, which suggests a role for interdomain association in the RBR ligase mechanism.

Interdomain communication revealed in the diabetes drug target mitoNEET

PubMed Central

Jennings, Patricia A.

2011-01-01

MitoNEET is a recently identified drug target for a commonly prescribed diabetes drug, Pioglitazone. It belongs to a previously uncharacterized ancient family of proteins for which the hallmark is the presence of a unique 39 amino acid CDGSH domain. In order to characterize the folding landscape of this novel fold, we performed thermodynamic simulations on MitoNEET using a structure-based model. Additionally, we implement a method of contact map clustering to partition out alternate pathways in folding. This cluster analysis reveals a detour late in folding and enables us to carefully examine the folding mechanism of each pathway rather than the macroscopic average. We observe that tightness in a region distal to the iron–sulfur cluster creates a constraint in folding and additionally appears to mediate communication in folding between the two domains of the protein. We demonstrate that by making changes at this site we are able to tweak the order of folding events in the cluster binding domain as well as decrease the barrier to folding. PMID:21402934

Database Resources of the BIG Data Center in 2018.

PubMed

2018-01-04

The BIG Data Center at Beijing Institute of Genomics (BIG) of the Chinese Academy of Sciences provides freely open access to a suite of database resources in support of worldwide research activities in both academia and industry. With the vast amounts of omics data generated at ever-greater scales and rates, the BIG Data Center is continually expanding, updating and enriching its core database resources through big-data integration and value-added curation, including BioCode (a repository archiving bioinformatics tool codes), BioProject (a biological project library), BioSample (a biological sample library), Genome Sequence Archive (GSA, a data repository for archiving raw sequence reads), Genome Warehouse (GWH, a centralized resource housing genome-scale data), Genome Variation Map (GVM, a public repository of genome variations), Gene Expression Nebulas (GEN, a database of gene expression profiles based on RNA-Seq data), Methylation Bank (MethBank, an integrated databank of DNA methylomes), and Science Wikis (a series of biological knowledge wikis for community annotations). In addition, three featured web services are provided, viz., BIG Search (search as a service; a scalable inter-domain text search engine), BIG SSO (single sign-on as a service; a user access control system to gain access to multiple independent systems with a single ID and password) and Gsub (submission as a service; a unified submission service for all relevant resources). All of these resources are publicly accessible through the home page of the BIG Data Center at http://bigd.big.ac.cn. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Slowing Translation between Protein Domains by Increasing Affinity between mRNAs and the Ribosomal Anti-Shine-Dalgarno Sequence Improves Solubility.

PubMed

Vasquez, Kevin A; Hatridge, Taylor A; Curtis, Nicholas C; Contreras, Lydia M

2016-02-19

Recent studies have demonstrated that effective protein production requires coordination of multiple cotranslational cellular processes, which are heavily affected by translation timing. Until recently, protein engineering has focused on codon optimization to maximize protein production rates, mostly considering the effect of tRNA abundance. However, as it relates to complex multidomain proteins, it has been hypothesized that strategic translational pauses between domains and between distinct individual structural motifs can prevent interactions between nascent chain fragments that generate kinetically trapped misfolded peptides and thereby enhance protein yields. In this study, we introduce synthetic transient pauses between structural domains in a heterologous model protein based on designed patterns of affinity between the mRNA and the anti-Shine-Dalgarno (aSD) sequence on the ribosome. We demonstrate that optimizing translation attenuation at domain boundaries can predictably affect solubility patterns in bacteria. Exploration of the affinity space showed that modifying less than 1% of the nucleotides (on a small 12 amino acid linker) can vary soluble protein yields up to ∼7-fold without altering the primary sequence of the protein. In the context of longer linkers, where a larger number of distinct structural motifs can fold outside the ribosome, optimal synonymous codon variations resulted in an additional 2.1-fold increase in solubility, relative to that of nonoptimized linkers of the same length. While rational construction of 54 linkers of various affinities showed a significant correlation between protein solubility and predicted affinity, only weaker correlations were observed between tRNA abundance and protein solubility. We also demonstrate that naturally occurring high-affinity clusters are present between structural domains of β-galactosidase, one of Escherichia coli's largest native proteins. Interdomain ribosomal affinity is an important factor that has not previously been explored in the context of protein engineering.

Unveiling Inherent Degeneracies in Determining Population-weighted Ensembles of Inter-domain Orientational Distributions Using NMR Residual Dipolar Couplings: Application to RNA Helix Junction Helix Motifs

PubMed Central

Yang, Shan; Al-Hashimi, Hashim M.

2016-01-01

A growing number of studies employ time-averaged experimental data to determine dynamic ensembles of biomolecules. While it is well known that different ensembles can satisfy experimental data to within error, the extent and nature of these degeneracies, and their impact on the accuracy of the ensemble determination remains poorly understood. Here, we use simulations and a recently introduced metric for assessing ensemble similarity to explore degeneracies in determining ensembles using NMR residual dipolar couplings (RDCs) with specific application to A-form helices in RNA. Various target ensembles were constructed representing different domain-domain orientational distributions that are confined to a topologically restricted (<10%) conformational space. Five independent sets of ensemble averaged RDCs were then computed for each target ensemble and a ‘sample and select’ scheme used to identify degenerate ensembles that satisfy RDCs to within experimental uncertainty. We find that ensembles with different ensemble sizes and that can differ significantly from the target ensemble (by as much as ΣΩ ~ 0.4 where ΣΩ varies between 0 and 1 for maximum and minimum ensemble similarity, respectively) can satisfy the ensemble averaged RDCs. These deviations increase with the number of unique conformers and breadth of the target distribution, and result in significant uncertainty in determining conformational entropy (as large as 5 kcal/mol at T = 298 K). Nevertheless, the RDC-degenerate ensembles are biased towards populated regions of the target ensemble, and capture other essential features of the distribution, including the shape. Our results identify ensemble size as a major source of uncertainty in determining ensembles and suggest that NMR interactions such as RDCs and spin relaxation, on their own, do not carry the necessary information needed to determine conformational entropy at a useful level of precision. The framework introduced here provides a general approach for exploring degeneracies in ensemble determination for different types of experimental data. PMID:26131693

Stimulation of Nipah Fusion: Small Intradomain Changes Trigger Extensive Interdomain Rearrangements.

PubMed

Dutta, Priyanka; Siddiqui, Ahnaf; Botlani, Mohsen; Varma, Sameer

2016-10-18

Nipah is an emerging paramyxovirus that is of serious concern to human health. It invades host cells using two of its membrane proteins-G and F. G binds to host ephrins and this stimulates G to activate F. Upon activation, F mediates virus-host membrane fusion. Here we focus on mechanisms that underlie the stimulation of G by ephrins. Experiments show that G interacts with ephrin and F through separate sites located on two different domains, the receptor binding domain (RBD) and the F activation domain (FAD). No models explain this allosteric coupling. In fact, the analogous mechanisms in other paramyxoviruses also remain undetermined. The structural organization of G is such that allosteric coupling must involve at least one of the two interfaces-the RBD-FAD interface and/or the RBD-RBD interface. Here we examine using molecular dynamics the effect of ephrin binding on the RBD-RBD interface. We find that despite inducing small changes in individual RBDs, ephrin reorients the RBD-RBD interface extensively, and in a manner that will enhance solvent exposure of the FAD. While this finding supports a proposed model of G stimulation, we also find from additional simulations that ephrin induces a similar RBD-RBD reorientation in a stimulation-deficient G mutant, V 209 VG → AAA. Together, our simulations suggest that while inter-RBD reorientation may be important, it is not, by itself, a sufficient condition for G stimulation. Additionally, we find that the mutation affects the conformational ensemble of RBD globally, including the RBD-FAD interface, suggesting the latter's role in G stimulation. Because ephrin induces small changes in individual RBDs, a proper analysis of conformational ensembles required that they are compared directly-we employ a method we developed recently, which we now release at SimTK, and show that it also performs excellently for non-Gaussian distributions. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Can single molecule localization microscopy be used to map closely spaced RGD nanodomains?

PubMed Central

Nicovich, Philip R.; Soeriyadi, Alexander; Nieves, Daniel J.; Gooding, J. Justin; Gaus, Katharina

2017-01-01

Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to detect nanodomains of the cell adhesion-stimulating tripeptide arginine-glycine-aspartic acid (RGD). These domains were formed by the phase separation of block copolymers with controllable spacing on the scale of tens of nanometers. We first determined the topology of the block copolymer with atomic force microscopy (AFM) and then imaged the localization of individual RGD peptides with direct stochastic optical reconstruction microscopy (dSTORM). To compare the data, we analyzed the dSTORM data with DBSCAN (density-based spatial clustering application with noise). The ligand distribution and polymer topology are not necessary identical since peptides may attach to the polymer outside the nanodomains and/or coupling and detection of peptides within the nanodomains is incomplete. We therefore performed simulations to explore the extent to which nanodomains could be mapped with dSTORM. We found that successful detection of nanodomains by dSTORM was influenced by the inter-domain spacing and the localization precision of individual fluorophores, and less by non-specific absorption of ligands to the substratum. For example, under our imaging conditions, DBSCAN identification of nanodomains spaced further than 50 nm apart was largely independent of background localisations, while nanodomains spaced closer than 50 nm required a localization precision of ~11 nm to correctly estimate the modal nearest neighbor distance (NDD) between nanodomains. We therefore conclude that SMLM is a promising technique to directly map the distribution and nanoscale organization of ligands and would benefit from an improved localization precision. PMID:28723958

The Effect of Shared Information on Pilot/Controller and Controller/Controller Interactions

NASA Technical Reports Server (NTRS)

Hansman, R. John; Davison, Hayley J.

2000-01-01

The increased ability to exchange information between Pilots, Controllers, Dispatchers, and other agents is a key component of advanced Air Traffic Management. The importance of shared information as well as current and evolving practices in information sharing are presented for a variety of interactions including: Controller/Pilot interactions, Pilot/Airline interactions, Controller/Controller interactions, and Airline/ATM interactions.

Structural studies of the natriuretic peptide receptor: a novel hormone-induced rotation mechanism for transmembrane signal transduction.

PubMed

Misono, Kunio S; Ogawa, Haruo; Qiu, Yue; Ogata, Craig M

2005-06-01

The atrial natriuretic peptide (ANP) receptor is a single-span transmembrane receptor that is coupled to its intrinsic intracellular guanylate cyclase (GCase) catalytic activity. To investigate the mechanisms of hormone binding and signal transduction, we have expressed the extracellular hormone-binding domain of the ANP receptor (ANPR) and characterized its structure and function. The disulfide-bond structure, state of glycosylation, binding-site residues, chloride-dependence of ANP binding, dimerization, and binding stoichiometry have been determined. More recently, the crystal structures of both the apoANPR dimer and ANP-bound complex have been determined. The structural comparison between the two has shown that, upon ANP binding, two ANPR molecules in the dimer undergo an inter-molecular twist with little intra-molecular conformational change. This motion produces a Ferris wheel-like translocation of two juxtamembrane domains with essentially no change in the inter-domain distance. This movement alters the relative orientation of the two domains equivalent to counter-clockwise rotation of each by 24 degrees . These results suggest that transmembrane signaling by the ANP receptor is mediated by a novel hormone-induced rotation mechanism.

A conserved π-cation and an electrostatic bridge are essential for 11R-lipoxygenase catalysis and structural stability.

PubMed

Eek, Priit; Piht, Mari-Ann; Rätsep, Margus; Freiberg, Arvi; Järving, Ivar; Samel, Nigulas

2015-10-01

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that consist of a regulatory calcium- and membrane-binding PLAT (polycystin-1, lipoxygenase, α-toxin) domain and a catalytic domain. In a previous study, the crystal structure of an 11R-LOX revealed a conserved π-cation bridge connecting these two domains which could mediate the regulatory effect of the PLAT domain to the active site. Here we analyzed the role of residues Trp107 and Lys172 that constitute the π-cation bridge in 11R-LOX along with Arg106 and Asp173-a potential salt bridge, which could also contribute to the inter-domain communication. According to our kinetic assays and protein unfolding experiments conducted using differential scanning fluorimetry and circular dichroism spectroscopy, mutants with a disrupted link display diminished catalytic activity alongside reduced stability of the protein fold. The results demonstrate that both these bridges contribute to the two-domain interface, and are important for proper enzyme activation. Copyright © 2015 Elsevier B.V. All rights reserved.

Crystal structure of a chimaeric bacterial glutamate dehydrogenase

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

Oliveira, Tânia; Sharkey, Michael A.; Engel, Paul C.

2016-05-23

Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P) +as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD +versusNADP +, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies,more » as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP +cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.« less

Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters

NASA Astrophysics Data System (ADS)

Landreh, Michael; Marklund, Erik G.; Uzdavinys, Povilas; Degiacomi, Matteo T.; Coincon, Mathieu; Gault, Joseph; Gupta, Kallol; Liko, Idlir; Benesch, Justin L. P.; Drew, David; Robinson, Carol V.

2017-01-01

Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.

Positional effects of fusion partners on the yield and solubility of MBP fusion proteins.

PubMed

Raran-Kurussi, Sreejith; Keefe, Karina; Waugh, David S

2015-06-01

Escherichia coli maltose-binding protein (MBP) is exceptionally effective at promoting the solubility of its fusion partners. However, there are conflicting reports in the literature claiming that (1) MBP is an effective solubility enhancer only when it is joined to the N-terminus of an aggregation-prone passenger protein, and (2) MBP is equally effective when fused to either end of the passenger. Here, we endeavor to resolve this controversy by comparing the solubility of a diverse set of MBP fusion proteins that, unlike those analyzed in previous studies, are identical in every way except for the order of the two domains. The results indicate that fusion proteins with an N-terminal MBP provide an excellent solubility advantage along with more robust expression when compared to analogous fusions in which MBP is the C-terminal fusion partner. We find that only intrinsically soluble passenger proteins (i.e., those not requiring a solubility enhancer) are produced as soluble fusions when they precede MBP. We also report that even subtle differences in inter-domain linker sequences can influence the solubility of fusion proteins. Published by Elsevier Inc.

Positional effects of fusion partners on the yield and solubility of MBP fusion proteins

PubMed Central

Raran-Kurussi, Sreejith; Keefe, Karina; Waugh, David S.

2015-01-01

Escherichia coli maltose-binding protein (MBP) is exceptionally effective at promoting the solubility of its fusion partners. However, there are conflicting reports in the literature claiming that 1) MBP is an effective solubility enhancer only when it is joined to the N-terminus of an aggregation-prone passenger protein, and 2) MBP is equally effective when fused to either end of the passenger. Here, we endeavor to resolve this controversy by comparing the solubility of a diverse set of MBP fusion proteins that, unlike those analyzed in previous studies, are identical in every way except for the order of the two domains. The results indicate that fusion proteins with an N-terminal MBP provide an excellent solubility advantage along with more robust expression when compared to analogous fusions in which MBP is the C-terminal fusion partner. We find that only intrinsically soluble passenger proteins (i.e., those not requiring a solubility enhancer) are produced as soluble fusions when they precede MBP. We also report that even subtle differences in inter-domain linker sequences can influence the solubility of fusion proteins. PMID:25782741

For a new look at 'lexical errors': evidence from semantic approximations with verbs in aphasia.

PubMed

Duvignau, Karine; Tran, Thi Mai; Manchon, Mélanie

2013-08-01

The ability to understand the similarity between two phenomena is fundamental for humans. Designated by the term analogy in psychology, this ability plays a role in the categorization of phenomena in the world and in the organisation of the linguistic system. The use of analogy in language often results in non-standard utterances, particularly in speakers with aphasia. These non-standard utterances are almost always studied in a nominal context and considered as errors. We propose a study of the verbal lexicon and present findings that measure, by an action-video naming task, the importance of verb-based non-standard utterances made by 17 speakers with aphasia ("la dame déshabille l'orange"/the lady undresses the orange, "elle casse la tomate"/she breaks the tomato). The first results we have obtained allow us to consider these type of utterances from a new perspective: we propose to eliminate the label of "error", suggesting that they may be viewed as semantic approximations based upon a relationship of inter-domain synonymy and are ingrained in the heart of the lexical system.

The cellulose binding region in Trichoderma reesei cellobiohydrolase I has a higher capacity in improving crystalline cellulose degradation than that of Penicillium oxalicum.

PubMed

Du, Jian; Zhang, Xiu; Li, Xuezhi; Zhao, Jian; Liu, Guodong; Gao, Baoyu; Qu, Yinbo

2018-06-19

Commercial cellulase preparations for lignocellulose bioconversion are mainly produced by the fungus Trichoderma reesei. The maximum cellulose conversion of T. reesei cellulase mixture was 15%-20% higher than that of Penicillium oxalicum in the hydrolysis of corncob residue and Avicel. Nevertheless, both preparations hydrolyzed more than 92% of cellulose in NaOH-mercerized Avicel. When added to Avicel hydrolysis residue that was less reactive to P. oxalicum cellulases, cellobiohydrolase I (CBH I) from T. reesei resulted in a higher cellulose conversion than its homologous proteins from P. oxalicum and Aspergillus niger at the same protein loadings. Further domain exchange experiment attributed the high hydrolytic efficiency of T. reesei CBH I to its inter-domain linker and cellulose-binding domain. The results in part explained the superior performance of T. reesei cellulases on the degradation of native crystalline cellulose, and highlighted the important role of cellulose-binding region in determining the degree of hydrolysis by cellulases. Copyright © 2018 Elsevier Ltd. All rights reserved.

Structural mechanism of ligand activation in human calcium-sensing receptor

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

Geng, Yong; Mosyak, Lidia; Kurinov, Igor

2016-07-19

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca 2+homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation.more » Our structures reveal multiple binding sites for Ca 2+and PO 4 3-ions. Both ions are crucial for structural integrity of the receptor. While Ca 2+ions stabilize the active state, PO 4 3-ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.« less

Capturing Three-Dimensional Genome Organization in Individual Cells by Single-Cell Hi-C.

PubMed

Nagano, Takashi; Wingett, Steven W; Fraser, Peter

2017-01-01

Hi-C is a powerful method to investigate genome-wide, higher-order chromatin and chromosome conformations averaged from a population of cells. To expand the potential of Hi-C for single-cell analysis, we developed single-cell Hi-C. Similar to the existing "ensemble" Hi-C method, single-cell Hi-C detects proximity-dependent ligation events between cross-linked and restriction-digested chromatin fragments in cells. A major difference between the single-cell Hi-C and ensemble Hi-C protocol is that the proximity-dependent ligation is carried out in the nucleus. This allows the isolation of individual cells in which nearly the entire Hi-C procedure has been carried out, enabling the production of a Hi-C library and data from individual cells. With this new method, we studied genome conformations and found evidence for conserved topological domain organization from cell to cell, but highly variable interdomain contacts and chromosome folding genome wide. In addition, we found that the single-cell Hi-C protocol provided cleaner results with less technical noise suggesting it could be used to improve the ensemble Hi-C technique.

Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function.

PubMed

Fischer, Audrey; Montal, Mauricio

2013-12-01

Clostridium botulinum neurotoxin (BoNT) is a multi-domain protein made up of the approximately 100 kDa heavy chain (HC) and the approximately 50 kDa light chain (LC). The HC can be further subdivided into two halves: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). We have investigated the minimal requirements for channel activity and LC translocation. We utilize a cellular protection assay and a single channel/single molecule LC translocation assay to characterize in real time the channel and chaperone activities of BoNT/A truncation constructs in Neuro 2A cells. The unstructured, elongated belt region of the TD is demonstrated to be dispensable for channel activity, although may be required for productive LC translocation. We show that the RBD is not necessary for channel activity or LC translocation, however it dictates the pH threshold of channel insertion into the membrane. These findings indicate that each domain functions as a chaperone for the others in addition to their individual functions, working in concert to achieve productive intoxication. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cost-effective handoff scheme based on mobility-aware dual pointer forwarding in proxy mobile IPv6 networks.

PubMed

Son, Seungsik; Jeong, Jongpil

2014-01-01

In this paper, a mobility-aware Dual Pointer Forwarding scheme (mDPF) is applied in Proxy Mobile IPv6 (PMIPv6) networks. The movement of a Mobile Node (MN) is classified as intra-domain and inter-domain handoff. When the MN moves, this scheme can reduce the high signaling overhead for intra-handoff/inter-handoff, because the Local Mobility Anchor (LMA) and Mobile Access Gateway (MAG) are connected by pointer chains. In other words, a handoff is aware of low mobility between the previously attached MAG (pMAG) and newly attached MAG (nMAG), and another handoff between the previously attached LMA (pLMA) and newly attached LMA (nLMA) is aware of high mobility. Based on these mobility-aware binding updates, the overhead of the packet delivery can be reduced. Also, we analyse the binding update cost and packet delivery cost for route optimization, based on the mathematical analytic model. Analytical results show that our mDPF outperforms the PMIPv6 and the other pointer forwarding schemes, in terms of reducing the total cost of signaling.

Biomarkers of a five-domain translational substrate for schizophrenia and schizoaffective psychosis.

PubMed

Fryar-Williams, Stephanie; Strobel, Jörg E

2015-01-01

The Mental Health Biomarker Project (2010-2014) selected commercial biochemistry markers related to monoamine synthesis and metabolism and measures of visual and auditory processing performance. Within a case-control discovery design with exclusion criteria designed to produce a highly characterised sample, results from 67 independently DSM IV-R-diagnosed cases of schizophrenia and schizoaffective disorder were compared with those from 67 control participants selected from a local hospital, clinic and community catchment area. Participants underwent protocol-based diagnostic-checking, functional-rating, biological sample-collection for thirty candidate markers and sensory-processing assessment. Fifteen biomarkers were identified on ROC analysis. Using these biomarkers, odds ratios, adjusted for a case-control design, indicated that schizophrenia and schizoaffective disorder were highly associated with dichotic listening disorder, delayed visual processing, low visual span, delayed auditory speed of processing, low reverse digit span as a measure of auditory working memory and elevated levels of catecholamines. Other nutritional and biochemical biomarkers were identified as elevated hydroxyl pyrroline-2-one as a marker of oxidative stress, vitamin D, B6 and folate deficits with elevation of serum B12 and free serum copper to zinc ratio. When individual biomarkers were ranked by odds ratio and correlated with clinical severity, five functional domains of visual processing, auditory processing, oxidative stress, catecholamines and nutritional-biochemical variables were formed. When the strengths of their inter-domain relationships were predicted by Lowess (non-parametric) regression, predominant bidirectional relationships were found between visual processing and catecholamine domains. At a cellular level, the nutritional-biochemical domain exerted a pervasive influence on the auditory domain as well as on all other domains. The findings of this biomarker research point towards a much-required advance in Psychiatry: quantification of some theoretically-understandable, translationally-informative, treatment-relevant underpinnings of serious mental illness. This evidence reveals schizophrenia and schizoaffective disorder in a somewhat different manner, as a conglomerate of several disorders many of which are not currently being assessed-for or treated in clinical settings. Currently available remediation techniques for these underlying conditions have potential to reduce treatment-resistance, relapse-prevention, cost burden and social stigma in these conditions. If replicated and validated in prospective trials, such findings will improve progress-monitoring and treatment-response for schizophrenia and schizoaffective disorder.

Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-beta signaling.

PubMed

Gao, Sheng; Alarcón, Claudio; Sapkota, Gopal; Rahman, Sadia; Chen, Pan-Yu; Goerner, Nina; Macias, Maria J; Erdjument-Bromage, Hediye; Tempst, Paul; Massagué, Joan

2009-11-13

TGF-beta induces phosphorylation of the transcription factors Smad2 and Smad3 at the C terminus as well as at an interdomain linker region. TGF-beta-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. Here, we identify Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain, Nedd4L specifically recognizes a TGF-beta-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 polyubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGF-beta-activated Smads and restricts the amplitude and duration of TGF-beta gene responses, and in mouse embryonic stem cells, it limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGF-beta signal transduction.

Identifying protein domains by global analysis of soluble fragment data.

PubMed

Bulloch, Esther M M; Kingston, Richard L

2014-11-15

The production and analysis of individual structural domains is a common strategy for studying large or complex proteins, which may be experimentally intractable in their full-length form. However, identifying domain boundaries is challenging if there is little structural information concerning the protein target. One experimental procedure for mapping domains is to screen a library of random protein fragments for solubility, since truncation of a domain will typically expose hydrophobic groups, leading to poor fragment solubility. We have coupled fragment solubility screening with global data analysis to develop an effective method for identifying structural domains within a protein. A gene fragment library is generated using mechanical shearing, or by uracil doping of the gene and a uracil-specific enzymatic digest. A split green fluorescent protein (GFP) assay is used to screen the corresponding protein fragments for solubility when expressed in Escherichia coli. The soluble fragment data are then analyzed using two complementary approaches. Fragmentation "hotspots" indicate possible interdomain regions. Clustering algorithms are used to group related fragments, and concomitantly predict domain location. The effectiveness of this Domain Seeking procedure is demonstrated by application to the well-characterized human protein p85α. Copyright © 2014 Elsevier Inc. All rights reserved.

Inherent flexibility of CLIC6 revealed by crystallographic and solution studies.

PubMed

Ferofontov, Alisa; Strulovich, Roi; Marom, Milit; Giladi, Moshe; Haitin, Yoni

2018-05-02

Chloride intracellular channels (CLICs) are a family of unique proteins, that were suggested to adopt both soluble and membrane-associated forms. Moreover, following this unusual metamorphic change, CLICs were shown to incorporate into membranes and mediate ion conduction in vitro, suggesting multimerization upon membrane insertion. Here, we present a 1.8 Å resolution crystal structure of the CLIC domain of mouse CLIC6 (mCLIC6). The structure reveals a monomeric arrangement and shows a high degree of structural conservation with other CLICs. Small-angle X-ray scattering (SAXS) analysis of mCLIC6 demonstrated that the overall solution structure is similar to the crystallographic conformation. Strikingly, further analysis of the SAXS data using ensemble optimization method unveiled additional elongated conformations, elucidating high structural plasticity as an inherent property of the protein. Moreover, structure-guided perturbation of the inter-domain interface by mutagenesis resulted in a population shift towards elongated conformations of mCLIC6. Additionally, we demonstrate that oxidative conditions induce an increase in mCLIC6 hydrophobicity along with mild oligomerization, which was enhanced by the presence of membrane mimetics. Together, these results provide mechanistic insights into the metamorphic nature of mCLIC6.

How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure.

PubMed

Wulff, Philip; Thomas, Claudia; Sargent, Frank; Armstrong, Fraser A

2016-03-01

'Oxygen-tolerant' [NiFe]-hydrogenases can catalyze H2 oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O2 molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H2 oxidation released at the active site of one partner are immediately transferred to its counterpart when an O2 molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O2-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins.

Using complementary approaches to identify trans-domain nuclear gene transfers in the extremophile Galdieria sulphuraria (Rhodophyta).

PubMed

Pandey, Ravi S; Saxena, Garima; Bhattacharya, Debashish; Qiu, Huan; Azad, Rajeev K

2017-02-01

Identification of horizontal gene transfers (HGTs) has primarily relied on phylogenetic tree based methods, which require a rich sampling of sequenced genomes to ensure a reliable inference. Because the success of phylogenetic approaches depends on the breadth and depth of the database, researchers usually apply stringent filters to detect only the most likely gene transfers in the genomes of interest. One such study focused on a highly conservative estimate of trans-domain gene transfers in the extremophile eukaryote, Galdieria sulphuraria (Galdieri) Merola (Rhodophyta), by applying multiple filters in their phylogenetic pipeline. This led to the identification of 75 inter-domain acquisitions from Bacteria or Archaea. Because of the evolutionary, ecological, and potential biotechnological significance of foreign genes in algae, alternative approaches and pipelines complementing phylogenetics are needed for a more comprehensive assessment of HGT. We present here a novel pipeline that uncovered 17 novel foreign genes of prokaryotic origin in G. sulphuraria, results that are supported by multiple lines of evidence including composition-based, comparative data, and phylogenetics. These genes encode a variety of potentially adaptive functions, from metabolite transport to DNA repair. © 2016 Phycological Society of America.

DNA binding triggers tetramerization of the glucocorticoid receptor in live cells

PubMed Central

Presman, Diego M.; Ganguly, Sourav; Schiltz, R. Louis; Johnson, Thomas A.; Karpova, Tatiana S.; Hager, Gordon L.

2016-01-01

Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. Although a large percentage of these proteins appear to self-associate to form dimers or higher order oligomers, the stoichiometry of DNA-bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligand-regulated transcription factor widely believed to act as a dimer or a monomer. Using a unique set of imaging techniques coupled with a cell line containing an array of DNA binding elements, we show that GR is predominantly a tetramer when bound to its target DNA. We find that DNA binding triggers an interdomain allosteric regulation within the GR, leading to tetramerization. We therefore propose that dynamic changes in GR stoichiometry represent a previously unidentified level of regulation in steroid receptor activation. Quaternary structure analysis of other members of the steroid receptor family (estrogen, androgen, and progesterone receptors) reveals variation in oligomerization states among this family of transcription factors. Because GR’s oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands and redefine the quaternary structure of steroid receptors. PMID:27382178

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

PubMed

Askin, Samuel P; Morin, Isabelle; Schaeffer, Patrick M

2011-08-15

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader. Copyright © 2011 Elsevier Inc. All rights reserved.

Highly conductive side chain block copolymer anion exchange membranes.

PubMed

Wang, Lizhu; Hickner, Michael A

2016-06-28

Block copolymers based on poly(styrene) having pendent trimethyl styrenylbutyl ammonium (with four carbon ring-ionic group alkyl linkers) or benzyltrimethyl ammonium groups with a methylene bridge between the ring and ionic group were synthesized by reversible addition-fragmentation radical (RAFT) polymerization as anion exchange membranes (AEMs). The C4 side chain polymer showed a 17% increase in Cl(-) conductivity of 33.7 mS cm(-1) compared to the benzyltrimethyl ammonium sample (28.9 mS cm(-1)) under the same conditions (IEC = 3.20 meq. g(-1), hydration number, λ = ∼7.0, cast from DMF/1-propanol (v/v = 3 : 1), relative humidity = 95%). As confirmed by small angle X-ray scattering (SAXS), the side chain block copolymers with tethered ammonium cations showed well-defined lamellar morphologies and a significant reduction in interdomain spacing compared to benzyltrimethyl ammonium containing block copolymers. The chemical stabilities of the block copolymers were evaluated under severe, accelerated conditions, and degradation was observed by (1)H NMR. The block copolymer with C4 side chain trimethyl styrenylbutyl ammonium motifs displayed slightly improved stability compared to that of a benzyltrimethyl ammonium-based AEM at 80 °C in 1 M NaOD aqueous solution for 30 days.

Molecular dynamics simulation unveils the conformational flexibility of the interdomain linker in the bacterial transcriptional regulator GabR from Bacillus subtilis bound to pyridoxal 5’-phosphate

PubMed Central

Narzi, Daniele; Guidoni, Leonardo

2017-01-01

GabR from Bacillus subtilis is a transcriptional regulator belonging to the MocR subfamily of the GntR regulators. The structure of the MocR regulators is characterized by the presence of two domains: i) a N-terminal domain, about 60 residue long, possessing the winged-Helix-Turn-Helix (wHTH) architecture with DNA recognition and binding capability; ii) a C-terminal domain (about 350 residue) folded as the pyridoxal 5’-phosphate (PLP) dependent aspartate aminotransferase (AAT) with dimerization and effector binding functions. The two domains are linked to each other by a peptide bridge. Although structural and functional characterization of MocRs is proceeding at a fast pace, virtually nothing is know about the molecular changes induced by the effector binding and on how these modifications influence the properties of the regulator. An extensive molecular dynamics simulation on the crystallographic structure of the homodimeric B. subtilis GabR has been undertaken with the aim to envisage the role and the importance of conformational flexibility in the action of GabR. Molecular dynamics has been calculated for the apo (without PLP) and holo (with PLP bound) forms of the GabR. A comparison between the molecular dynamics trajectories calculated for the two GabR forms suggested that one of the wHTH domain detaches from the AAT-like domain in the GabR PLP-bound form. The most evident conformational change in the holo PLP-bound form is represented by the rotation and the subsequent detachment from the subunit surface of one of the wHTH domains. The movement is mediated by a rearrangement of the linker connecting the AAT domain possibly triggered by the presence of the negative charge of the PLP cofactor. This is the second most significant conformational modification. The C-terminal section of the linker docks into the “active site” pocket and establish stabilizing contacts consisting of hydrogen-bonds, salt-bridges and hydrophobic interactions. PMID:29253008

Pathogenicity Determinants of the Human Malaria Parasite Plasmodium falciparum Have Ancient Origins

PubMed Central

Brazier, Andrew J.; Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell

2017-01-01

ABSTRACT Plasmodium falciparum, the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi. We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum. Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum-infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum, it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P. falciparum. Our findings suggest that parasite adhesion traits associated with both mild and severe malaria have much earlier origins than previously appreciated and have important implications for virulence evolution in a major human pathogen. PMID:28101534

Pathogenicity Determinants of the Human Malaria Parasite Plasmodium falciparum Have Ancient Origins.

PubMed

Brazier, Andrew J; Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell; Smith, Joseph D

2017-01-01

Plasmodium falciparum , the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi . We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum . Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum -infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum , it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P. falciparum . Our findings suggest that parasite adhesion traits associated with both mild and severe malaria have much earlier origins than previously appreciated and have important implications for virulence evolution in a major human pathogen.

Interaction Control to Synchronize Non-synchronizable Networks.

PubMed

Schröder, Malte; Chakraborty, Sagar; Witthaut, Dirk; Nagler, Jan; Timme, Marc

2016-11-17

Synchronization constitutes one of the most fundamental collective dynamics across networked systems and often underlies their function. Whether a system may synchronize depends on the internal unit dynamics as well as the topology and strength of their interactions. For chaotic units with certain interaction topologies synchronization might be impossible across all interaction strengths, meaning that these networks are non-synchronizable. Here we propose the concept of interaction control, generalizing transient uncoupling, to induce desired collective dynamics in complex networks and apply it to synchronize even such non-synchronizable systems. After highlighting that non-synchronizability prevails for a wide range of networks of arbitrary size, we explain how a simple binary control may localize interactions in state space and thereby synchronize networks. Intriguingly, localizing interactions by a fixed control scheme enables stable synchronization across all connected networks regardless of topological constraints. Interaction control may thus ease the design of desired collective dynamics even without knowledge of the networks' exact interaction topology and consequently have implications for biological and self-organizing technical systems.

Interaction Control to Synchronize Non-synchronizable Networks

PubMed Central

Schröder, Malte; Chakraborty, Sagar; Witthaut, Dirk; Nagler, Jan; Timme, Marc

2016-01-01

Synchronization constitutes one of the most fundamental collective dynamics across networked systems and often underlies their function. Whether a system may synchronize depends on the internal unit dynamics as well as the topology and strength of their interactions. For chaotic units with certain interaction topologies synchronization might be impossible across all interaction strengths, meaning that these networks are non-synchronizable. Here we propose the concept of interaction control, generalizing transient uncoupling, to induce desired collective dynamics in complex networks and apply it to synchronize even such non-synchronizable systems. After highlighting that non-synchronizability prevails for a wide range of networks of arbitrary size, we explain how a simple binary control may localize interactions in state space and thereby synchronize networks. Intriguingly, localizing interactions by a fixed control scheme enables stable synchronization across all connected networks regardless of topological constraints. Interaction control may thus ease the design of desired collective dynamics even without knowledge of the networks’ exact interaction topology and consequently have implications for biological and self-organizing technical systems. PMID:27853266

Shifts in a fish's resource holding power during a contact paired interaction: the influence of a copper-contaminated diet in rainbow trout.

PubMed

Campbell, H A; Handy, R D; Sims, D W

2005-01-01

The influence of sublethal chronic dietary copper (Cu) exposure on the dominant-subordinate relationship between pairs of juvenile rainbow trout (Oncorhynchus mykiss) was examined. Fish were fed either a normal (11 mg Cu kg(-1) food) or Cu-contaminated (721 mg Cu kg(-1) food) diet for 8 wk. Paired interactions were observed--control versus control, Cu-exposed versus Cu-exposed, and control versus Cu-exposed fish--using a computer-aided video tracking system to measure duration of interactions, total distance moved, and the number of encounters during each contest. In concurrence with game theory, each interaction became escalated with a lesser size disparity between contestants. However, in Cu-exposed versus Cu-exposed fish interactions, the dominant-subordinate relationship was decided sooner and with less aggression than a control versus control fish interaction with fish of a similar relative body mass disparity. During control versus Cu-exposed fish interactions, control fish would normally dominate interactions (12 out of 16 bouts) unless the Cu-exposed fish had a 15% body mass advantage. Muscle glycogen and lactate levels after each contest reflected the duration of bouts and winners of the contests, irrespective of Cu exposure. We conclude that Cu-contaminated fish are less able to compete and have lower resource holding power than controls and will withdraw from a contest at a lower level of interaction, unless a size advantage in the Cu-exposed fish increases the probability of winning.

Molecular Simulations of Mutually Exclusive Folding in a Two-Domain Protein Switch

PubMed Central

Mills, Brandon M.; Chong, Lillian T.

2011-01-01

A major challenge with testing designs of protein conformational switches is the need for experimental probes that can independently monitor their individual protein domains. One way to circumvent this issue is to use a molecular simulation approach in which each domain can be directly observed. Here we report what we believe to be the first molecular simulations of mutually exclusive folding in an engineered two-domain protein switch, providing a direct view of how folding of one protein drives unfolding of the other in a barnase-ubiquitin fusion protein. These simulations successfully capture the experimental effects of interdomain linker length and ligand binding on the extent of unfolding in the less stable domain. In addition, the effect of linker length on the potential for oligomerization, which eliminates switch activity, is in qualitative agreement with analytical ultracentrifugation experiments. We also perform what we believe to be the first study of protein unfolding via progressive localized compression. Finally, we are able to explore the kinetics of mutually exclusive folding by determining the effect of linker length on rates of unfolding and refolding of each protein domain. Our results demonstrate that molecular simulations can provide seemingly novel biological insights on the behavior of individual protein domains, thereby aiding in the rational design of bifunctional switches. PMID:21281591

Multidomain flavin-dependent sulfhydryl oxidases.

PubMed

Coppock, Donald L; Thorpe, Colin

2006-01-01

Eukaryotic flavin-dependent sulfhydryl oxidases catalyze oxidative protein folding with the generation of disulfides and the reduction of oxygen to hydrogen peroxide. This review deals principally with the Quiescinsulfhydryl oxidases (QSOX) that are found in multiple forms in multicellular organisms and singly in a number of protozoan parasites. QSOX is an ancient fusion of thioredoxin domains and an FAD-binding module, ERV1/ALR. Interdomain disulfide exchanges transmit reducing equivalents from substrates to the flavin cofactor and thence to molecular oxygen. The in vitro substrate specificity of avian QSOX1 and the likely substrates of QSOXs in vivo are discussed. The location of QSOX immunoreactivity and mRNA expression levels in human cells and tissues is reviewed. Generally, there is a marked association of QSOX1 expression with cell types that have a high secretory load of disulfide-containing peptides and proteins. The abundance of sulfhydryl oxidases in the islets of Langerhans suggests that oxidative protein folding may directly contribute to the oxidative stress believed to be a factor in the progression to type II diabetes. Finally, the structure and mechanism of QSOX proteins is compared to their smaller stand-alone cousins: yeast ERV1p and ERV2p, the mammalian augmenter of liver regeneration (ALR), and the viral ALR homologs.

Membrane-Induced Structural Rearrangement and Identification of a Novel Membrane Anchor in Talin F2F3

PubMed Central

Arcario, Mark J.; Tajkhorshid, Emad

2014-01-01

Experimental challenges associated with characterization of the membrane-bound form of talin have prevented us from understanding the molecular mechanism of its membrane-dependent integrin activation. Here, utilizing what we believe to be a novel membrane mimetic model, we present a reproducible model of membrane-bound talin observed across multiple independent simulations. We characterize both local and global membrane-induced structural transitions that successfully reconcile discrepancies between biochemical and structural studies and provide insight into how talin might modulate integrin function. Membrane binding of talin, captured in unbiased simulations, proceeds through three distinct steps: initial electrostatic recruitment of the F2 subdomain to anionic lipids via several basic residues; insertion of an initially buried, conserved hydrophobic anchor into the membrane; and association of the F3 subdomain with the membrane surface through a large, interdomain conformational change. These latter two steps, to our knowledge, have not been observed or described previously. Electrostatic analysis shows talin F2F3 to be highly polarized, with a highly positive underside, which we attribute to the initial electrostatic recruitment, and a negative top face, which can help orient the protein optimally with respect to the membrane, thereby reducing the number of unproductive membrane collision events. PMID:25418091

A concept ideation framework for medical device design.

PubMed

Hagedorn, Thomas J; Grosse, Ian R; Krishnamurty, Sundar

2015-06-01

Medical device design is a challenging process, often requiring collaboration between medical and engineering domain experts. This collaboration can be best institutionalized through systematic knowledge transfer between the two domains coupled with effective knowledge management throughout the design innovation process. Toward this goal, we present the development of a semantic framework for medical device design that unifies a large medical ontology with detailed engineering functional models along with the repository of design innovation information contained in the US Patent Database. As part of our development, existing medical, engineering, and patent document ontologies were modified and interlinked to create a comprehensive medical device innovation and design tool with appropriate properties and semantic relations to facilitate knowledge capture, enrich existing knowledge, and enable effective knowledge reuse for different scenarios. The result is a Concept Ideation Framework for Medical Device Design (CIFMeDD). Key features of the resulting framework include function-based searching and automated inter-domain reasoning to uniquely enable identification of functionally similar procedures, tools, and inventions from multiple domains based on simple semantic searches. The significance and usefulness of the resulting framework for aiding in conceptual design and innovation in the medical realm are explored via two case studies examining medical device design problems. Copyright © 2015 Elsevier Inc. All rights reserved.

Phylogeny of Rieske/cytb Complexes with a Special Focus on the Haloarchaeal Enzymes

PubMed Central

Baymann, Frauke; Schoepp-Cothenet, Barbara; Lebrun, Evelyne; van Lis, Robert; Nitschke, Wolfgang

2012-01-01

Rieske/cytochrome b (Rieske/cytb) complexes are proton pumping quinol oxidases that are present in most bacteria and Archaea. The phylogeny of their subunits follows closely the 16S-rRNA phylogeny, indicating that chemiosmotic coupling was already present in the last universal common ancestor of Archaea and bacteria. Haloarchaea are the only organisms found so far that acquired Rieske/cytb complexes via interdomain lateral gene transfer. They encode two Rieske/cytb complexes in their genomes; one of them is found in genetic context with nitrate reductase genes and has its closest relatives among Actinobacteria and the Thermus/Deinococcus group. It is likely to function in nitrate respiration. The second Rieske/cytb complex of Haloarchaea features a split cytochrome b sequence as do Cyanobacteria, chloroplasts, Heliobacteria, and Bacilli. It seems that Haloarchaea acquired this complex from an ancestor of the above-mentioned phyla. Its involvement in the bioenergetic reaction chains of Haloarchaea is unknown. We present arguments in favor of the hypothesis that the ancestor of Haloarchaea, which relied on a highly specialized bioenergetic metabolism, that is, methanogenesis, and was devoid of quinones and most enzymes of anaerobic or aerobic bioenergetic reaction chains, integrated laterally transferred genes into its genome to respond to a change in environmental conditions that made methanogenesis unfavorable. PMID:22798450

Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

PubMed

Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

2016-12-13

Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

Determination of the rotational diffusion tensor of macromolecules in solution from nmr relaxation data with a combination of exact and approximate methods--application to the determination of interdomain orientation in multidomain proteins.

PubMed

Ghose, R; Fushman, D; Cowburn, D

2001-04-01

In this paper we present a method for determining the rotational diffusion tensor from NMR relaxation data using a combination of approximate and exact methods. The approximate method, which is computationally less intensive, computes values of the principal components of the diffusion tensor and estimates the Euler angles, which relate the principal axis frame of the diffusion tensor to the molecular frame. The approximate values of the principal components are then used as starting points for an exact calculation by a downhill simplex search for the principal components of the tensor over a grid of the space of Euler angles relating the diffusion tensor frame to the molecular frame. The search space of Euler angles is restricted using the tensor orientations calculated using the approximate method. The utility of this approach is demonstrated using both simulated and experimental relaxation data. A quality factor that determines the extent of the agreement between the measured and predicted relaxation data is provided. This approach is then used to estimate the relative orientation of SH3 and SH2 domains in the SH(32) dual-domain construct of Abelson kinase complexed with a consolidated ligand. Copyright 2001 Academic Press.

Mechanism-Based Mathematical Model for Gating of Ionotropic Glutamate Receptors.

PubMed

Dai, Jian; Wollmuth, Lonnie P; Zhou, Huan-Xiang

2015-08-27

We present a mathematical model for ionotropic glutamate receptors (iGluR's) that is built on mechanistic understanding and yields a number of thermodynamic and kinetic properties of channel gating. iGluR's are ligand-gated ion channels responsible for the vast majority of fast excitatory neurotransmission in the central nervous system. The effects of agonist-induced closure of the ligand-binding domain (LBD) are transmitted to the transmembrane channel (TMC) via interdomain linkers. Our model demonstrates that, relative to full agonists, partial agonists may reduce either the degree of LBD closure or the curvature of the LBD free energy basin, leading to less stabilization of the channel open state and hence lower channel open probability. A rigorous relation is derived between the channel closed-to-open free energy difference and the tension within the linker. Finally, by treating LBD closure and TMC opening as diffusive motions, we obtain gating trajectories that resemble stochastic current traces from single-channel recordings and calculate the rate constants for transitions between the channel open and closed states. Our model can be implemented by molecular dynamics simulations to realistically depict iGluR gating and may guide functional experiments in gaining deeper insight into this essential family of channel proteins.

Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

DOE PAGES

Alberstein, Robert; Grey, Richard; Zimmet, Austin; ...

2015-10-12

Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysismore » reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. In this paper, we hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and finally suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.« less

3D Graphene-Ni Foam as an Advanced Electrode for High-Performance Nonaqueous Redox Flow Batteries.

PubMed

Lee, Kyubin; Lee, Jungkuk; Kwon, Kyoung Woo; Park, Min-Sik; Hwang, Jin-Ha; Kim, Ki Jae

2017-07-12

Electrodes composed of multilayered graphene grown on a metal foam (GMF) were prepared by directly growing multilayer graphene sheets on a three-dimensional (3D) Ni-foam substrate via a self-catalyzing chemical vapor deposition process. The multilayer graphene sheets are successfully grown on the Ni-foam substrate surface, maintaining the unique 3D macroporous structure of the Ni foam. The potential use of GMF electrodes in nonaqueous redox flow batteries (RFBs) is carefully examined using [Co(bpy) 3 ] +/2+ and [Fe(bpy) 3 ] 2+/3+ redox couples. The GMF electrodes display a much improved electrochemical activity and enhanced kinetics toward the [Co(bpy) 3 ] +/2+ (anolyte) and [Fe(bpy) 3 ] 2+/3+ (catholyte) redox couples, compared with the bare Ni metal foam electrodes, suggesting that the 2D graphene sheets having lots of interdomain defects provide sufficient reaction sites and secure electric-conduction pathways. Consequently, a nonaqueous RFB cell assembled with GMF electrodes exhibits high Coulombic and voltage efficiencies of 87.2 and 90.9%, respectively, at the first cycle. This performance can be maintained up to the 50th cycle without significant efficiency loss. Moreover, the importance of a rational electrode design for improving electrochemical performance is addressed.

Protein domain assignment from the recurrence of locally similar structures

PubMed Central

Tai, Chin-Hsien; Sam, Vichetra; Gibrat, Jean-Francois; Garnier, Jean; Munson, Peter J.

2010-01-01

Domains are basic units of protein structure and essential for exploring protein fold space and structure evolution. With the structural genomics initiative, the number of protein structures in the Protein Databank (PDB) is increasing dramatically and domain assignments need to be done automatically. Most existing structural domain assignment programs define domains using the compactness of the domains and/or the number and strength of intra-domain versus inter-domain contacts. Here we present a different approach based on the recurrence of locally similar structural pieces (LSSPs) found by one-against-all structure comparisons with a dataset of 6,373 protein chains from the PDB. Residues of the query protein are clustered using LSSPs via three different procedures to define domains. This approach gives results that are comparable to several existing programs that use geometrical and other structural information explicitly. Remarkably, most of the proteins that contribute the LSSPs defining a domain do not themselves contain the domain of interest. This study shows that domains can be defined by a collection of relatively small locally similar structural pieces containing, on average, four secondary structure elements. In addition, it indicates that domains are indeed made of recurrent small structural pieces that are used to build protein structures of many different folds as suggested by recent studies. PMID:21287617

Cognitive Correlates of Metamemory in Alzheimer's Disease

PubMed Central

Shaked, Danielle; Farrell, Meagan; Huey, Edward; Metcalfe, Janet; Cines, Sarah; Karlawish, Jason; Sullo, Elisabeth; Cosentino, Stephanie

2014-01-01

Objective Metamemory, or knowledge of one's memory abilities, is often impaired in individuals with Alzheimer's disease (AD), although the basis of this metacognitive deficit has not been fully articulated. Behavioral and imaging studies have produced conflicting evidence regarding the extent to which specific cognitive domains (i.e., executive functioning (EF), memory) and brain regions contribute to memory awareness. The primary aim of this study was to disentangle the cognitive correlates of metamemory in AD by examining the relatedness of objective metamemory performance to cognitive tasks grouped by domain (EF or memory) as well as by preferential hemispheric reliance defined by task modality (verbal or nonverbal). Method 89 participants with mild AD recruited at Columbia University Medical Center and the University of Pennsylvania underwent objective metamemory and cognitive testing. Partial correlations were used to assess the relationship between metamemory and four cognitive variables, adjusted for recruitment site. Results The significant correlates of metamemory included nonverbal fluency (r = .27 p = .02) and nonverbal memory (r = .24, p = .04). Conclusions Our findings suggest that objectively measured metamemory in a large sample of individuals with mild AD is selectively related to a set of inter-domain nonverbal tasks. The association between metamemory and the nonverbal tasks may implicate a shared reliance on a right-sided cognitive network that spans frontal and temporal regions. PMID:24819066

Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design.

PubMed

Akerboom, Jasper; Rivera, Jonathan D Vélez; Guilbe, María M Rodríguez; Malavé, Elisa C Alfaro; Hernandez, Hector H; Tian, Lin; Hires, S Andrew; Marvin, Jonathan S; Looger, Loren L; Schreiter, Eric R

2009-03-06

The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca2+-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.

Evolution of EF-hand calcium-modulated proteins. IV. Exon shuffling did not determine the domain compositions of EF-hand proteins

NASA Technical Reports Server (NTRS)

Kretsinger, R. H.; Nakayama, S.

1993-01-01

In the previous three reports in this series we demonstrated that the EF-hand family of proteins evolved by a complex pattern of gene duplication, transposition, and splicing. The dendrograms based on exon sequences are nearly identical to those based on protein sequences for troponin C, the essential light chain myosin, the regulatory light chain, and calpain. This validates both the computational methods and the dendrograms for these subfamilies. The proposal of congruence for calmodulin, troponin C, essential light chain, and regulatory light chain was confirmed. There are, however, significant differences in the calmodulin dendrograms computed from DNA and from protein sequences. In this study we find that introns are distributed throughout the EF-hand domain and the interdomain regions. Further, dendrograms based on intron type and distribution bear little resemblance to those based on protein or on DNA sequences. We conclude that introns are inserted, and probably deleted, with relatively high frequency. Further, in the EF-hand family exons do not correspond to structural domains and exon shuffling played little if any role in the evolution of this widely distributed homolog family. Calmodulin has had a turbulent evolution. Its dendrograms based on protein sequence, exon sequence, 3'-tail sequence, intron sequences, and intron positions all show significant differences.

Determination of the Rotational Diffusion Tensor of Macromolecules in Solution from NMR Relaxation Data with a Combination of Exact and Approximate Methods—Application to the Determination of Interdomain Orientation in Multidomain Proteins

NASA Astrophysics Data System (ADS)

Ghose, Ranajeet; Fushman, David; Cowburn, David

2001-04-01

In this paper we present a method for determining the rotational diffusion tensor from NMR relaxation data using a combination of approximate and exact methods. The approximate method, which is computationally less intensive, computes values of the principal components of the diffusion tensor and estimates the Euler angles, which relate the principal axis frame of the diffusion tensor to the molecular frame. The approximate values of the principal components are then used as starting points for an exact calculation by a downhill simplex search for the principal components of the tensor over a grid of the space of Euler angles relating the diffusion tensor frame to the molecular frame. The search space of Euler angles is restricted using the tensor orientations calculated using the approximate method. The utility of this approach is demonstrated using both simulated and experimental relaxation data. A quality factor that determines the extent of the agreement between the measured and predicted relaxation data is provided. This approach is then used to estimate the relative orientation of SH3 and SH2 domains in the SH(32) dual-domain construct of Abelson kinase complexed with a consolidated ligand.

Novel Analysis of Oceanic Surface Water Metagenomes Suggests Importance of Polyphosphate Metabolism in Oligotrophic Environments

PubMed Central

Temperton, Ben; Gilbert, Jack A.; Quinn, John P.; McGrath, John W.

2011-01-01

Polyphosphate is a ubiquitous linear homopolymer of phosphate residues linked by high-energy bonds similar to those found in ATP. It has been associated with many processes including pathogenicity, DNA uptake and multiple stress responses across all domains. Bacteria have also been shown to use polyphosphate as a way to store phosphate when transferred from phosphate-limited to phosphate-rich media – a process exploited in wastewater treatment and other environmental contaminant remediation. Despite this, there has, to date, been little research into the role of polyphosphate in the survival of marine bacterioplankton in oligotrophic environments. The three main proteins involved in polyphosphate metabolism, Ppk1, Ppk2 and Ppx are multi-domain and have differential inter-domain and inter-gene conservation, making unbiased analysis of relative abundance in metagenomic datasets difficult. This paper describes the development of a novel Isofunctional Homolog Annotation Tool (IHAT) to detect homologs of genes with a broad range of conservation without bias of traditional expect-value cutoffs. IHAT analysis of the Global Ocean Sampling (GOS) dataset revealed that genes associated with polyphosphate metabolism are more abundant in environments where available phosphate is limited, suggesting an important role for polyphosphate metabolism in marine oligotrophs. PMID:21305044

Control Surface Interaction Effects of the Active Aeroelastic Wing Wind Tunnel Model

NASA Technical Reports Server (NTRS)

Heeg, Jennifer

2006-01-01

This paper presents results from testing the Active Aeroelastic Wing wind tunnel model in NASA Langley s Transonic Dynamics Tunnel. The wind tunnel test provided an opportunity to study aeroelastic system behavior under combined control surface deflections, testing for control surface interaction effects. Control surface interactions were observed in both static control surface actuation testing and dynamic control surface oscillation testing. The primary method of evaluating interactions was examination of the goodness of the linear superposition assumptions. Responses produced by independently actuating single control surfaces were combined and compared with those produced by simultaneously actuating and oscillating multiple control surfaces. Adjustments to the data were required to isolate the control surface influences. Using dynamic data, the task increases, as both the amplitude and phase have to be considered in the data corrections. The goodness of static linear superposition was examined and analysis of variance was used to evaluate significant factors influencing that goodness. The dynamic data showed interaction effects in both the aerodynamic measurements and the structural measurements.

A study of interactive control scheduling and economic assessment for robotic systems

NASA Technical Reports Server (NTRS)

1982-01-01

A class of interactive control systems is derived by generalizing interactive manipulator control systems. Tasks of interactive control systems can be represented as a network of a finite set of actions which have specific operational characteristics and specific resource requirements, and which are of limited duration. This has enabled the decomposition of the overall control algorithm simultaneously and asynchronously. The performance benefits of sensor referenced and computer-aided control of manipulators in a complex environment is evaluated. The first phase of the CURV arm control system software development and the basic features of the control algorithms and their software implementation are presented. An optimal solution for a production scheduling problem that will be easy to implement in practical situations is investigated.

Mono and Multivalency In Tethered Protein-Carbohydrate Bonds

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

Ratto, T V; Langry, K C; Rudd, R E

2004-01-29

Molecular recognition in biological systems typically involves large numbers of interactions simultaneously. By using a multivalent approach, weak interactions with fairly low specificity can become strong highly specific interactions. Additionally, this allows an organism to control the strength and specificity of an interaction simply by controlling the number of binding molecules (or binding sites), which in turn can be controlled through transcriptional regulation.

Effect of interaction strength on robustness of controlling edge dynamics in complex networks

NASA Astrophysics Data System (ADS)

Pang, Shao-Peng; Hao, Fei

2018-05-01

Robustness plays a critical role in the controllability of complex networks to withstand failures and perturbations. Recent advances in the edge controllability show that the interaction strength among edges plays a more important role than network structure. Therefore, we focus on the effect of interaction strength on the robustness of edge controllability. Using three categories of all edges to quantify the robustness, we develop a universal framework to evaluate and analyze the robustness in complex networks with arbitrary structures and interaction strengths. Applying our framework to a large number of model and real-world networks, we find that the interaction strength is a dominant factor for the robustness in undirected networks. Meanwhile, the strongest robustness and the optimal edge controllability in undirected networks can be achieved simultaneously. Different from the case of undirected networks, the robustness in directed networks is determined jointly by the interaction strength and the network's degree distribution. Moreover, a stronger robustness is usually associated with a larger number of driver nodes required to maintain full control in directed networks. This prompts us to provide an optimization method by adjusting the interaction strength to optimize the robustness of edge controllability.

Reduction of Helicopter Blade-Vortex Interaction Noise by Active Rotor Control Technology

NASA Technical Reports Server (NTRS)

Yu, Yung H.; Gmelin, Bernd; Splettstoesser, Wolf; Brooks, Thomas F.; Philippe, Jean J.; Prieur, Jean

1997-01-01

Helicopter blade-vortex interaction noise is one of the most severe noise sources and is very important both in community annoyance and military detection. Research over the decades has substantially improved basic physical understanding of the mechanisms generating rotor blade-vortex interaction noise and also of controlling techniques, particularly using active rotor control technology. This paper reviews active rotor control techniques currently available for rotor blade vortex interaction noise reduction, including higher harmonic pitch control, individual blade control, and on-blade control technologies. Basic physical mechanisms of each active control technique are reviewed in terms of noise reduction mechanism and controlling aerodynamic or structural parameters of a blade. Active rotor control techniques using smart structures/materials are discussed, including distributed smart actuators to induce local torsional or flapping deformations, Published by Elsevier Science Ltd.

Experiment and simulation for CSI: What are the missing links?

NASA Technical Reports Server (NTRS)

Belvin, W. Keith; Park, K. C.

1989-01-01

Viewgraphs on experiment and simulation for control structure interaction (CSI) are presented. Topics covered include: control structure interaction; typical control/structure interaction system; CSI problem classification; actuator/sensor models; modeling uncertainty; noise models; real-time computations; and discrete versus continuous.

Cooperative airframe/propulsion control for supersonic cruise aircraft

NASA Technical Reports Server (NTRS)

Schweikhard, W. G.; Berry, D. T.

1974-01-01

Interactions between propulsion systems and flight controls have emerged as a major control problem on supersonic cruise aircraft. This paper describes the nature and causes of these interactions and the approaches to predicting and solving the problem. Integration of propulsion and flight control systems appears to be the most promising solution if the interaction effects can be adequately predicted early in the vehicle design. Significant performance, stability, and control improvements may be realized from a cooperative control system.

The Interaction Between Punishment Sensitivity and Effortful Control for Emerging Adults' Substance Use Behaviors.

PubMed

Kahn, Rachel E; Chiu, Pearl H; Deater-Deckard, Kirby; Hochgraf, Anna K; King-Casas, Brooks; Kim-Spoon, Jungmeen

2018-01-08

Within the dual systems perspective, high reward sensitivity and low punishment sensitivity in conjunction with deficits in cognitive control may contribute to high levels of risk taking, such as substance use. The current study examined whether the individual components of effortful control (inhibitory control, attentional control, and activation control) serve as regulators and moderate the association between reward or punishment sensitivity and substance use behaviors. A total of 1,808 emerging adults from a university setting (Mean age = 19.48; 72% female) completed self-report measures of reward and punishment sensitivity, effortful control, and substance use. Findings indicated significant two-way interactions for punishment sensitivity and inhibitory control for alcohol and marijuana use. The form of these interactions revealed a significant negative association between punishment sensitivity and alcohol and marijuana use at low levels of inhibitory control. No significant interactions emerged for reward sensitivity or other components of effortful control. The current findings provide preliminary evidence suggesting the dual systems theorized to influence risk taking behavior interact to make joint contributions to health risk behaviors such as substance use in emerging adults.

Structural Dynamics and Control Interaction of Flexible Structures

NASA Technical Reports Server (NTRS)

Ryan, Robert S. (Editor); Scofield, Harold N. (Editor)

1987-01-01

A workshop on structural dynamics and control interaction of flexible structures was held to promote technical exchange between the structural dynamics and control disciplines, foster joint technology, and provide a forum for discussing and focusing critical issues in the separate and combined areas. Issues and areas of emphasis were identified in structure-control interaction for the next generation of flexible systems.

Synthesis and applications of heterostructured semiconductor nanocrystals

NASA Astrophysics Data System (ADS)

Khon, Elena

Semiconductor nanocrystals (NCs) have been of great interest to researchers for several decades due to their unique optoelectronic properties. These nanoparticles are widely used for a variety of different applications. However, there are many unresolved issues that lower the efficiency and/or stability of devices which incorporate these NCs. Our research is dedicated to addressing these issues by identifying potential problems and resolving them, improving existing systems, generating new synthetic strategies, and/or building new devices. The general strategies for the synthesis of different nanocrystals were established in this work, one of which is the colloidal growth of gold domains onto CdS semiconductor nanocrystals. Control of shape and size was achieved simply by adjusting the temperature and the time of the reaction. Depending on the exact morphology of Au and CdS domains, fabricated nano-composites can undergo evaporation-induced self-assembly onto a substrate, which is very useful for building devices. CdS/Au heterostructures can assemble in two different ways: through end-to-end coupling of Au domains, resulting in the formation of one-dimensional chains; and via side-by-side packing of CdS nanorods, leading to the onset of two-dimensional superlattices. We investigated the nature of exciton-plasmon interactions in Au-tipped CdS nanorods using femtosecond transient absorption spectroscopy. The study demonstrated that the key optoelectronic properties of electrically coupled metal and semiconductor domains are significantly different from those observed in systems with weak inter-domain coupling. In particular, strongly-coupled nanocomposites promote mixing of electronic states at semiconductor-metal domain interfaces, which causes a significant suppression of both plasmon and exciton carrier excitations. Colloidal QDs are starting to replace organic molecules in many different applications, such as organic light emmiting diods (OLEDs), due to their light emmision tunability. We reported a general strategy for the assembly of all-inorganic light-emitting nanocrystal films with an emission quantum yield in the 30-52% range. Our methodology relies on solution-processing of CdSe nanocrystals into a crystalline matrix of a wide band gap semiconductor (CdS, ZnS). As a result, we replace original organic ligands on nanocrystal surfaces with an inorganic medium which efficiently preserves the quantum confinement of electrical charges in CdSe NCs. In addition to strong emission, fabricated films demonstrated excellent thermal and chemical stability, and a large refractive index, which avails their integration into emerging solid-state nanocrystal devices. The ability to control size and shape of NCs is essential as it automatically affects the optoelectronic properties of the crystals. Colloidal chemistry offers an assortment of synthetic tools for tuning the shape of NCs, but some nanoparticle morphologies require alternative processing strategies. We have shown that chemical etching of colloidal nanoparticles can facilitate the realization of desirable nanocrystal geometries. This methodology allows both CdSe and CdS composed semiconductor domains be exposed to the external environment, while maintaining a structural design that is highly desirable for catalytic applications. Hydrogen production tests confirmed the improved catalytic activity of CdSe/CdS dimers. We expect that the demonstrated application will become a common methodology in the synthesis of charge-separating nanocrystals, leading to advanced nanoparticle architectures for applications in the areas of photocatalysis, photovoltaics, and light detection.

Microgrid Controls | Grid Modernization | NREL

Science.gov Websites

Systems Integration Facility. Microgrid Controller Interaction with Distribution Management Systems This project investigates the interaction of distribution management systems with local controllers, including microgrid controllers. The project is developing integrated control and management systems for distribution

Interactive computer graphics and its role in control system design of large space structures

NASA Technical Reports Server (NTRS)

Reddy, A. S. S. R.

1985-01-01

This paper attempts to show the relevance of interactive computer graphics in the design of control systems to maintain attitude and shape of large space structures to accomplish the required mission objectives. The typical phases of control system design, starting from the physical model such as modeling the dynamics, modal analysis, and control system design methodology are reviewed and the need of the interactive computer graphics is demonstrated. Typical constituent parts of large space structures such as free-free beams and free-free plates are used to demonstrate the complexity of the control system design and the effectiveness of the interactive computer graphics.

Use of electronic games by young children and fundamental movement skills?

PubMed

Barnett, Lisa M; Hinkley, Trina; Okely, Anthony D; Hesketh, Kylie; Salmon, Jo

2012-06-01

This study investigated associations between pre-school children's time spent playing electronic games and their fundamental movement skills. In 2009, 53 children had physical activity (Actigraph accelerometer counts per minute), parent proxy-report of child's time in interactive and non-interactive electronic games (min./week), and movement skill (Test of Gross Motor Development-2) assessed. Hierarchical linear regression, adjusting for age (range = 3-6 years), sex (Step 1), and physical activity (cpm; M=687, SD=175.42; Step 2), examined the relationship between time in (a) non-interactive and (b) interactive electronic games and locomotor and object control skill. More than half (59%, n=31) of the children were female. Adjusted time in interactive game use was associated with object control but not locomotor skill. Adjusted time in non-interactive game use had no association with object control or locomotor skill. Greater time spent playing interactive electronic games is associated with higher object control skill proficiency in these young children. Longitudinal and experimental research is required to determine if playing these games improves object control skills or if children with greater object control skill proficiency prefer and play these games.

Interactive Television: The Influence of User Control and Interactive Structure.

ERIC Educational Resources Information Center

Gagnon, Diana; And Others

A series of studies underway at the Audience Research Facility at MIT (the Massachusetts Institute of Technology) are examining the influence of interactive video on learning and entertainment television viewing. The first study compared the learning of spatial content from interactive (user controlled video games) versus observational…

Visual analytics of geo-social interaction patterns for epidemic control.

PubMed

Luo, Wei

2016-08-10

Human interaction and population mobility determine the spatio-temporal course of the spread of an airborne disease. This research views such spreads as geo-social interaction problems, because population mobility connects different groups of people over geographical locations via which the viruses transmit. Previous research argued that geo-social interaction patterns identified from population movement data can provide great potential in designing effective pandemic mitigation. However, little work has been done to examine the effectiveness of designing control strategies taking into account geo-social interaction patterns. To address this gap, this research proposes a new framework for effective disease control; specifically this framework proposes that disease control strategies should start from identifying geo-social interaction patterns, designing effective control measures accordingly, and evaluating the efficacy of different control measures. This framework is used to structure design of a new visual analytic tool that consists of three components: a reorderable matrix for geo-social mixing patterns, agent-based epidemic models, and combined visualization methods. With real world human interaction data in a French primary school as a proof of concept, this research compares the efficacy of vaccination strategies between the spatial-social interaction patterns and the whole areas. The simulation results show that locally targeted vaccination has the potential to keep infection to a small number and prevent spread to other regions. At some small probability, the local control strategies will fail; in these cases other control strategies will be needed. This research further explores the impact of varying spatial-social scales on the success of local vaccination strategies. The results show that a proper spatial-social scale can help achieve the best control efficacy with a limited number of vaccines. The case study shows how GS-EpiViz does support the design and testing of advanced control scenarios in airborne disease (e.g., influenza). The geo-social patterns identified through exploring human interaction data can help target critical individuals, locations, and clusters of locations for disease control purposes. The varying spatial-social scales can help geographically and socially prioritize limited resources (e.g., vaccines).

Interactions between butterfly-shaped pulses in the inhomogeneous media

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

Liu, Wen-Jun; Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190; Huang, Long-Gang

2014-10-15

Pulse interactions affect pulse qualities during the propagation. Interactions between butterfly-shaped pulses are investigated to improve pulse qualities in the inhomogeneous media. In order to describe the interactions between butterfly-shaped pulses, analytic two-soliton solutions are derived. Based on those solutions, influences of corresponding parameters on pulse interactions are discussed. Methods to control the pulse interactions are suggested. - Highlights: • Interactions between butterfly-shaped pulses are investigated. • Methods to control the pulse interactions are suggested. • Analytic two-soliton solutions for butterfly-shaped pulses are derived.

How do emotion and motivation direct executive control?

PubMed

Pessoa, Luiz

2009-04-01

Emotion and motivation have crucial roles in determining human behavior. Yet, how they interact with cognitive control functions is less understood. Here, the basic elements of a conceptual framework for understanding how they interact are introduced. More broadly, the 'dual competition' framework proposes that emotion and motivation affect both perceptual and executive competition. In particular, the anterior cingulate cortex is hypothesized to be engaged in attentional/effortful control mechanisms and to interact with several other brain structures, including the amygdala and nucleus accumbens, in integrating affectively significant signals with control signals in prefrontal cortex. An implication of the proposal is that emotion and motivation can either enhance or impair behavioral performance depending on how they interact with control functions.

Mother-child interactions in depressed children and children at high risk and low risk for future depression.

PubMed

Dietz, Laura J; Birmaher, Boris; Williamson, Douglas E; Silk, Jennifer S; Dahl, Ronald E; Axelson, David A; Ehmann, Mary; Ryan, Neal D

2008-05-01

To compare mother-child interactions and parenting styles in families of children with major depressive disorder, youths at high risk for depression, and healthy controls. Currently depressed (n = 43), high-risk (n = 28), and healthy control (n = 41) youths and their mothers engaged in a standardized videotaped problem-solving interaction. Measures of affect and behavior for both mothers and children were obtained, in addition to global measures of parenting. Depressed children demonstrated more negativity and less positivity in dyadic interactions than did children at high risk and control children. Mothers of depressed children were more disengaged than control mothers. Exploratory repeated-measures analyses in a subgroup of depressed children (n = 16) suggested mother-child interactions do not significantly change when children recover from depression. Children at high risk demonstrated less positivity in dyadic interactions than did controls. Mothers with a history of major depressive disorder and mothers with higher current depressive symptoms demonstrated patterns of disengagement and low control in interactions with children. Mother-child interactions in depressed youths are marked by maternal disengagement and low child positivity that may not improve when children recover. The bidirectional effects of maternal disengagement and low levels of child positivity may precede onset of major depressive disorder in children and serve as risk factors for recurrent depression in youths.

Team Electronic Gameplay Combining Different Means of Control

NASA Technical Reports Server (NTRS)

Palsson, Olafur S. (Inventor); Pope, Alan T. (Inventor)

2014-01-01

Disclosed are methods and apparatuses provided for modifying the effect of an operator controlled input device on an interactive device to encourage the self-regulation of at least one physiological activity by a person different than the operator. The interactive device comprises a display area which depicts images and apparatus for receiving at least one input from the operator controlled input device to thus permit the operator to control and interact with at least some of the depicted images. One effect modification comprises measurement of the physiological activity of a person different from the operator, while modifying the ability of the operator to control and interact with at least some of the depicted images by modifying the input from the operator controlled input device in response to changes in the measured physiological signal.

Functional, interactive and critical health literacy: Varying relationships with control over care and number of GP visits.

PubMed

van der Heide, Iris; Heijmans, Monique; Schuit, A Jantine; Uiters, Ellen; Rademakers, Jany

2015-08-01

The aim of this study is to examine the extent to which functional, interactive and critical health literacy are associated with patients' perceived control over care and frequency of GP visits. Data from the Dutch 'National Panel of People with Chronic Illness or Disability' was used (N=2508). Health literacy was assessed by the Functional, Communicative and Critical Health Literacy measure. Perceived control over care was indicated by perceived ability to organize care, interact with providers and to perform self-care. By multivariate linear and logistic regression analyses, associations between health literacy and perceived control over care and subsequently frequency of GP visits were studied. Mainly interactive health literacy was associated with patients' perceived ability to organize care, interact with healthcare providers and perform self-care, whereas only functional health literacy was associated with number of GP visits. The results imply that some patients' may be less able to exert control over their care because of lower health literacy. Functional, interactive and critical health literacy vary in their relevance for patients' ability to exert control. Initiatives for strengthening patients' role in healthcare may be improved by paying attention to patients' health literacy, specifically functional and interactive health literacy. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Examining relations between locus of control, loneliness, subjective well-being, and preference for online social interaction.

PubMed

Ye, Yinghua; Lin, Lin

2015-02-01

The unprecedented popularity of online communication has raised interests and concerns among the public as well as in scholarly circles. Online communications have pushed people farther away from one another. This study is a further examination of the effects of online communications on well-being, in particular: Locus of control, Loneliness, Subjective well-being, and Preference for online social interaction. Chinese undergraduate students (N = 260; 84 men, 176 women; M age = 20.1 yr., SD = 1.2) were questioned about demographic information and use of social media as well as four previously validated questionnaires related to well-being. Most participants used QQ, a popular social networking program, as the major channel for online social interactions. Locus of control was positively related to Loneliness and Preference for online social interaction, but negatively related to Subjective well-being; Loneliness (positively) and Subjective well-being (negatively) were related to Preference for online social interaction; and Loneliness and Subjective well-being had a full mediating effect between the relationships of Locus of control and Preference for online social interaction. The findings of the study showed that more lonely, unhappy, and externally controlled students were more likely to be engaged in online social interaction. Improving students' locus of control, loneliness, and happiness may help reduce problematic Internet use.

Human performance interfaces in air traffic control.

PubMed

Chang, Yu-Hern; Yeh, Chung-Hsing

2010-01-01

This paper examines how human performance factors in air traffic control (ATC) affect each other through their mutual interactions. The paper extends the conceptual SHEL model of ergonomics to describe the ATC system as human performance interfaces in which the air traffic controllers interact with other human performance factors including other controllers, software, hardware, environment, and organisation. New research hypotheses about the relationships between human performance interfaces of the system are developed and tested on data collected from air traffic controllers, using structural equation modelling. The research result suggests that organisation influences play a more significant role than individual differences or peer influences on how the controllers interact with the software, hardware, and environment of the ATC system. There are mutual influences between the controller-software, controller-hardware, controller-environment, and controller-organisation interfaces of the ATC system, with the exception of the controller-controller interface. Research findings of this study provide practical insights in managing human performance interfaces of the ATC system in the face of internal or external change, particularly in understanding its possible consequences in relation to the interactions between human performance factors.

Adaptive Controller Effects on Pilot Behavior

NASA Technical Reports Server (NTRS)

Trujillo, Anna C.; Gregory, Irene M.; Hempley, Lucas E.

2014-01-01

Adaptive control provides robustness and resilience for highly uncertain, and potentially unpredictable, flight dynamics characteristic. Some of the recent flight experiences of pilot-in-the-loop with an adaptive controller have exhibited unpredicted interactions. In retrospect, this is not surprising once it is realized that there are now two adaptive controllers interacting, the software adaptive control system and the pilot. An experiment was conducted to categorize these interactions on the pilot with an adaptive controller during control surface failures. One of the objectives of this experiment was to determine how the adaptation time of the controller affects pilots. The pitch and roll errors, and stick input increased for increasing adaptation time and during the segment when the adaptive controller was adapting. Not surprisingly, altitude, cross track and angle deviations, and vertical velocity also increase during the failure and then slowly return to pre-failure levels. Subjects may change their behavior even as an adaptive controller is adapting with additional stick inputs. Therefore, the adaptive controller should adapt as fast as possible to minimize flight track errors. This will minimize undesirable interactions between the pilot and the adaptive controller and maintain maneuvering precision.

Domain organization of p130, PLC-related catalytically inactive protein, and structural basis for the lack of enzyme activity.

PubMed

Kanematsu, T; Yoshimura, K; Hidaka, K; Takeuchi, H; Katan, M; Hirata, M

2000-05-01

The 130-kDa protein (p130) was isolated as a novel inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]-binding protein similar to phospholipase C-delta1 (PLC-delta1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. & Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. & Hirata, M. (1996) Biochem. J. 313, 319-325]. To test experimentally the domain organization of p130 and structural basis for lack of PLC activity, we subjected p130 to limited proteolysis and also constructed a number of chimeras with PLC-delta1. Trypsin treatment of p130 produced four major polypeptides with molecular masses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were calculated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC-delta1 and demonstrating that all domains of p130, including the unique region at the C-terminus, are stable, tightly folded structures. p130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that p130 has intrinsically no PLC activity. A number of chimeric molecules between p130 and PLC-delta1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two proteins, as only some domains of p130 could replace the corresponding structures in PLC-delta1 to form a functional enzyme. These results suggest that p130 and the related proteins could represent a new protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.

Synergistic interaction between job control and social support at work on depression, burnout, and insomnia among Japanese civil servants.

PubMed

Saijo, Yasuaki; Chiba, Shigeru; Yoshioka, Eiji; Nakagi, Yoshihiko; Ito, Toshihiro; Kitaoka-Higashiguchi, Kazuyo; Yoshida, Takahiko

2015-02-01

To elucidate whether low job control and low social support at work have synergistic interaction on mental health. The synergistic interaction was also analyzed after stratification by high and low job demands. Participants were 2,121 local government employees in Asahikawa city, Japan. The Brief Job Stress Questionnaire was used to assess job demands, job control, and social support. Depression was assessed using the Patient Health Questionnaire-9. The Maslach Burnout Inventory-General Survey was used to assess burnout. Insomnia was assessed using the Athens Insomnia Scale. Possible confounder-adjusted logistic regression analyses were performed to obtain odds ratios for depression, burnout, and insomnia, and synergy indices between job control and social support at work were assessed. The synergy indices among men and women, respectively, were 2.08 (80 % confidence interval: 1.01, 4.27) and 1.98 (0.67, 5.89) for depression, 1.79 (1.28, 2.51) and 2.62 (1.07, 6.40) for burnout, and 1.92 (1.22, 3.02) and 2.77 (0.43, 18.01) for insomnia. Men with high job demands had higher synergistic interaction on depression and burnout, compared to men with low job demands, and women with low job demands had higher synergistic interaction between job control and social support at work on burnout and insomnia, compared to women with high job demands. There were more-than-additive interactions of job control and social support at work on depression, burnout, and insomnia. After stratification by job demands, the synergistic interaction may be different between men and women. To assess job stress, it is necessary to consider the interactive effect of not only job demands and job control but also job control and social support at work.

Affiliation and control in marital interaction: interpersonal complementarity is present but is not associated with affect or relationship quality.

PubMed

Cundiff, Jenny M; Smith, Timothy W; Butner, Jonathan; Critchfield, Kenneth L; Nealey-Moore, Jill

2015-01-01

The principle of complementarity in interpersonal theory states that an actor's behavior tends to "pull, elicit, invite, or evoke" responses from interaction partners who are similar in affiliation (i.e., warmth vs. hostility) and opposite in control (i.e., dominance vs. submissiveness). Furthermore, complementary interactions are proposed to evoke less negative affect and promote greater relationship satisfaction. These predictions were examined in two studies of married couples. Results suggest that complementarity in affiliation describes a robust general pattern of marital interaction, but complementarity in control varies across contexts. Consistent with behavioral models of marital interaction, greater levels of affiliation and lower control by partners-not complementarity in affiliation or control-were associated with less anger and anxiety and greater relationship quality. Partners' levels of affiliation and control combined in ways other than complementarity-mostly additively, but sometimes synergistically-to predict negative affect and relationship satisfaction. © 2014 by the Society for Personality and Social Psychology, Inc.

Modeling pilot interaction with automated digital avionics systems: Guidance and control algorithms for contour and nap-of-the-Earth flight

NASA Technical Reports Server (NTRS)

Hess, Ronald A.

1990-01-01

A collection of technical papers are presented that cover modeling pilot interaction with automated digital avionics systems and guidance and control algorithms for contour and nap-of-the-earth flight. The titles of the papers presented are as follows: (1) Automation effects in a multiloop manual control system; (2) A qualitative model of human interaction with complex dynamic systems; (3) Generalized predictive control of dynamic systems; (4) An application of generalized predictive control to rotorcraft terrain-following flight; (5) Self-tuning generalized predictive control applied to terrain-following flight; and (6) Precise flight path control using a predictive algorithm.

Human-computer interface including haptically controlled interactions

DOEpatents

Anderson, Thomas G.

2005-10-11

The present invention provides a method of human-computer interfacing that provides haptic feedback to control interface interactions such as scrolling or zooming within an application. Haptic feedback in the present method allows the user more intuitive control of the interface interactions, and allows the user's visual focus to remain on the application. The method comprises providing a control domain within which the user can control interactions. For example, a haptic boundary can be provided corresponding to scrollable or scalable portions of the application domain. The user can position a cursor near such a boundary, feeling its presence haptically (reducing the requirement for visual attention for control of scrolling of the display). The user can then apply force relative to the boundary, causing the interface to scroll the domain. The rate of scrolling can be related to the magnitude of applied force, providing the user with additional intuitive, non-visual control of scrolling.

Unconditional analyses can increase efficiency in assessing gene-environment interaction of the case-combined-control design.

PubMed

Goldstein, Alisa M; Dondon, Marie-Gabrielle; Andrieu, Nadine

2006-08-01

A design combining both related and unrelated controls, named the case-combined-control design, was recently proposed to increase the power for detecting gene-environment (GxE) interaction. Under a conditional analytic approach, the case-combined-control design appeared to be more efficient and feasible than a classical case-control study for detecting interaction involving rare events. We now propose an unconditional analytic strategy to further increase the power for detecting gene-environment (GxE) interactions. This strategy allows the estimation of GxE interaction and exposure (E) main effects under certain assumptions (e.g. no correlation in E between siblings and the same exposure frequency in both control groups). Only the genetic (G) main effect cannot be estimated because it is biased. Using simulations, we show that unconditional logistic regression analysis is often more efficient than conditional analysis for detecting GxE interaction, particularly for a rare gene and strong effects. The unconditional analysis is also at least as efficient as the conditional analysis when the gene is common and the main and joint effects of E and G are small. Under the required assumptions, the unconditional analysis retains more information than does the conditional analysis for which only discordant case-control pairs are informative leading to more precise estimates of the odds ratios.

How do emotion and motivation direct executive control?

PubMed Central

Pessoa, Luiz

2009-01-01

Emotion and motivation have crucial roles in determining human behavior. Yet, how they interact with cognitive control functions is less understood. Here, the basic elements of a conceptual framework for understanding how they interact are introduced. More broadly, the `dual competition' framework proposes that emotion and motivation affect both perceptual and executive competition. In particular, the anterior cingulate cortex is hypothesized to be engaged in attentional/effortful control mechanisms and to interact with several other brain structures, including the amygdala and nucleus accumbens, in integrating affectively significant signals with control signals in prefrontal cortex. An implication of the proposal is that emotion and motivation can either enhance or impair behavioral performance depending on how they interact with control functions. PMID:19285913

A walking prescription for statically-stable walkers based on walker/terrain interaction

NASA Technical Reports Server (NTRS)

Nagy, Peter V.; Whittaker, William L.; Desa, Subhas

1992-01-01

The walker/terrain interaction phenomena for the control of a statically stable walking machine are described. The algorithms, measures, and knowledge of walker/terrain interaction phenomena are then combined to form a prescription for how to walk on general terrain. This prescription consists of two parts: nominal control and reactive control. The function of nominal control is the evaluation and execution of planned motions, based on predicted foot force redistributions, to achieve reliable locomotion. The function of reactive control is the monitoring of walker/terrain interaction in real-time to detect anomalous conditions and then respond with the appropriate reflexive actions. Simulations and experiments have been used to test and verify various aspects of the walking prescription.

Human Factors Considerations in System Design

NASA Technical Reports Server (NTRS)

Mitchell, C. M. (Editor); Vanbalen, P. M. (Editor); Moe, K. L. (Editor)

1983-01-01

Human factors considerations in systems design was examined. Human factors in automated command and control, in the efficiency of the human computer interface and system effectiveness are outlined. The following topics are discussed: human factors aspects of control room design; design of interactive systems; human computer dialogue, interaction tasks and techniques; guidelines on ergonomic aspects of control rooms and highly automated environments; system engineering for control by humans; conceptual models of information processing; information display and interaction in real time environments.

Tracking control of time-varying knee exoskeleton disturbed by interaction torque.

PubMed

Li, Zhan; Ma, Wenhao; Yin, Ziguang; Guo, Hongliang

2017-11-01

Knee exoskeletons have been increasingly applied as assistive devices to help lower-extremity impaired people to make their knee joints move through providing external movement compensation. Tracking control of knee exoskeletons guided by human intentions often encounters time-varying (time-dependent) issues and the disturbance interaction torque, which may dramatically put an influence up on their dynamic behaviors. Inertial and viscous parameters of knee exoskeletons can be estimated to be time-varying due to unexpected mechanical vibrations and contact interactions. Moreover, the interaction torque produced from knee joint of wearers has an evident disturbance effect on regular motions of knee exoskeleton. All of these points can increase difficultly of accurate control of knee exoskeletons to follow desired joint angle trajectories. This paper proposes a novel control strategy for controlling knee exoskeleton with time-varying inertial and viscous coefficients disturbed by interaction torque. Such designed controller is able to make the tracking error of joint angle of knee exoskeletons exponentially converge to zero. Meanwhile, the proposed approach is robust to guarantee the tracking error bounded when the interaction torque exists. Illustrative simulation and experiment results are presented to show efficiency of the proposed controller. Additionally, comparisons with gradient dynamic (GD) approach and other methods are also presented to demonstrate efficiency and superiority of the proposed control strategy for tracking joint angle of knee exoskeleton. Copyright © 2017 ISA. Published by Elsevier Ltd. All rights reserved.

Effects of Pilates exercises on sensory interaction, postural control and fatigue in patients with multiple sclerosis.

PubMed

Soysal Tomruk, Melda; Uz, Muhammed Zahid; Kara, Bilge; İdiman, Egemen

2016-05-01

Decreased postural control, sensory integration deficits and fatigue are important problems that cause functional impairments in patients with multiple sclerosis (pwMS). To examine the effect of modified clinical Pilates exercises on sensory interaction and balance, postural control and fatigue in pwMS. Eleven patients with multiple sclerosis and 12 healthy matched controls were recruited in this study. Limits of stability and postural stability tests were used to evaluate postural control by Biodex Balance System and sensory interaction assessed. Fatigue was assessed by Modified Fatigue Impact Scale. Pilates exercises were applied two times a week for 10 weeks and measurements were repeated to pwMS after exercise training. Postural control and fatigue (except psychosocial parameter) of pwMS were significantly worser than healthy controls (p<0.05). Significant improvements occurred in sensory interaction (eyes open, foam surface) and total, physical and cognitive scores of fatigue after 10-week modified clinical Pilates training (p<0.05). No significant changes were detected in postural control after the pilates exercises (p>0.05). Ten-week Pilates training is effective to improve sensory interaction and to decrease fatigue. Pilates exercises can be applied safely in ambulatory pwMS for enhance sensory interaction and balance and combat fatigue. More investigations are needed. Copyright © 2016 Elsevier B.V. All rights reserved.

The NASA controls-structures interaction technology program

NASA Technical Reports Server (NTRS)

Newsom, Jerry R.; Layman, W. E.; Waites, H. B.; Hayduk, R. J.

1990-01-01

The interaction between a flexible spacecraft structure and its control system is commonly referred to as controls-structures interaction (CSI). The CSI technology program is developing the capability and confidence to integrate the structure and control system, so as to avoid interactions that cause problems and to exploit interactions to increase spacecraft capability. A NASA program has been initiated to advance CSI technology to a point where it can be used in spacecraft design for future missions. The CSI technology program is a multicenter program utilizing the resources of the NASA Langley Research Center (LaRC), the NASA Marshall Space Flight Center (MSFC), and the NASA Jet Propulsion Laboratory (JPL). The purpose is to describe the current activities, results to date, and future activities of the NASA CSI technology program.

Psychological control in daily parent-child interactions increases children's negative emotions.

PubMed

Aunola, Kaisa; Tolvanen, Asko; Viljaranta, Jaana; Nurmi, Jari-Erik

2013-06-01

The aim of the present study was to investigate the temporal dynamics between parental behaviors in daily interactions with their offspring, that is, affection and psychological control, and children's negative emotions. The participants were 152 Finnish families with a 6- to 7-year-old child. Children's negative emotions and parental affection and psychological control in interactions with their child were measured daily using diary questionnaires filled in by the mothers and fathers over 7 successive days. The results of multilevel modeling showed that psychological control applied by mothers and fathers in daily interactions with their child leads to an increase in negative emotions in the child. Parental affection in daily interactions with their child was not associated with the child's negative emotions. PsycINFO Database Record (c) 2013 APA, all rights reserved.

Interactions between soil thermal and hydrological dynamics in the response of Alaska ecosystems to fire disturbance

Treesearch

Shuhua Yi; David McGuire; Jennifer Harden; Eric Kasischke; Kristen Manies; Larr Hinzman; Anna Liljedahl; Jim Randerson; Heping Liu; Vladimire Romanovsky; Sergei Marchenko; Yongwon Kim

2009-01-01

Soil temperature and moisture are important factors that control many ecosystem processes. However, interactions between soil thermal and hydrological processes are not adequately understood in cold regions, where the frozen soil, fire disturbance, and soil drainage play important roles in controlling interactions among these processes. These interactions were...

Mapping Control and Affiliation in Teacher-Student Interaction with State Space Grids

ERIC Educational Resources Information Center

Mainhard, M. Tim; Pennings, Helena J. M.; Wubbels, Theo; Brekelmans, Mieke

2012-01-01

This paper explores how State Space Grids (SSG), a dynamic systems research method, can be used to map teacher-student interactions from moment-to-moment and thereby to incorporate temporal aspects of interaction. Interactions in two secondary school classrooms are described in terms of level of interpersonal control and affiliation, and of…

Micromechanical Devices for Control of Cell-Cell Interaction, and Methods of Use Thereof

NASA Technical Reports Server (NTRS)

Bhatia, Sangeeta N. (Inventor); Hui, Elliot (Inventor)

2017-01-01

The development and function of living tissues depends largely on interactions between cells that can vary in both time and space; however, temporal control of cell-cell interaction is experimentally challenging. By employing a micromachined silicon substrate with moving parts, herein is disclosed the dynamic regulation of cell-cell interactions via direct manipulation of adherent cells with micron-scale precision. The inventive devices and methods allow mechanical control of both tissue composition and spatial organization. The inventive device and methods enable the investigation of dynamic cell-cell interaction in a multitude of applications, such as intercellular communication, spanning embryogenesis, homeostasis, and pathogenic processes.

Study design options in evaluating gene-environment interactions: practical considerations for a planned case-control study of pediatric leukemia.

PubMed

Goodman, Michael; Dana Flanders, W

2007-04-01

We compare methodological approaches for evaluating gene-environment interaction using a planned study of pediatric leukemia as a practical example. We considered three design options: a full case-control study (Option I), a case-only study (Option II), and a partial case-control study (Option III), in which information on controls is limited to environmental exposure only. For each design option we determined its ability to measure the main effects of environmental factor E and genetic factor G, and the interaction between E and G. Using the leukemia study example, we calculated sample sizes required to detect and odds ratio (OR) of 2.0 for E alone, an OR of 10 for G alone and an interaction G x E of 3. Option I allows measuring both main effects and interaction, but requires a total sample size of 1,500 cases and 1,500 controls. Option II allows measuring only interaction, but requires just 121 cases. Option III allows calculating the main effect of E, and interaction, but not the main effect of G, and requires a total of 156 cases and 133 controls. In this case, the partial case-control study (Option III) appears to be more efficient with respect to its ability to answer the research questions for the amount of resources required. The design options considered in this example are not limited to observational epidemiology and may be applicable in studies of pharmacogenomics, survivorship, and other areas of pediatric ALL research.

Interaction between telencephalic signals and respiratory dynamics in songbirds

PubMed Central

Méndez, Jorge M.; Mindlin, Gabriel B.

2012-01-01

The mechanisms by which telencephalic areas affect motor activities are largely unknown. They could either take over motor control from downstream motor circuits or interact with the intrinsic dynamics of these circuits. Both models have been proposed for telencephalic control of respiration during learned vocal behavior in birds. The interactive model postulates that simple signals from the telencephalic song control areas are sufficient to drive the nonlinear respiratory network into producing complex temporal sequences. We tested this basic assumption by electrically stimulating telencephalic song control areas and analyzing the resulting respiratory patterns in zebra finches and in canaries. We found strong evidence for interaction between the rhythm of stimulation and the intrinsic respiratory rhythm, including naturally emerging subharmonic behavior and integration of lateralized telencephalic input. The evidence for clear interaction in our experimental paradigm suggests that telencephalic vocal control also uses a similar mechanism. Furthermore, species differences in the response of the respiratory system to stimulation show parallels to differences in the respiratory patterns of song, suggesting that the interactive production of respiratory rhythms is manifested in species-specific specialization of the involved circuitry. PMID:22402649

Proceedings of the Workshop on Identification and Control of Flexible Space Structures, volume 1

NASA Technical Reports Server (NTRS)

Rodriguez, G. (Editor)

1985-01-01

Identification and control of flexible space structures were studied. Exploration of the most advanced modeling estimation, identification and control methodologies to flexible space structures was discussed. The following general areas were discussed: space platforms, antennas, and flight experiments; control/structure interactions - modeling, integrated design and optimization, control and stabilization, and shape control; control technology; control of space stations; large antenna control, dynamics and control experiments, and control/structure interaction experiments.

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

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening

NASA Astrophysics Data System (ADS)

Kalid, Ori; Mense, Martin; Fischman, Sharon; Shitrit, Alina; Bihler, Hermann; Ben-Zeev, Efrat; Schutz, Nili; Pedemonte, Nicoletta; Thomas, Philip J.; Bridges, Robert J.; Wetmore, Diana R.; Marantz, Yael; Senderowitz, Hanoch

2010-12-01

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism.

PubMed

Summers, Emma L; Cumming, Mathew H; Oulavallickal, Tifany; Roberts, Nicholas J; Arcus, Vickery L

2017-08-01

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an -apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co-factor bound in the active site. A central hinge region creates a "butterfly-like" motion of the domains that reduces the width of the inter-domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life. © 2017 The Protein Society.

A new structural framework for integrating replication protein A into DNA processing machinery

PubMed Central

Brosey, Chris A.; Yan, Chunli; Tsutakawa, Susan E.; Heller, William T.; Rambo, Robert P.; Tainer, John A.; Ivanov, Ivaylo; Chazin, Walter J.

2013-01-01

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA’s DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA’s DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways. PMID:23303776

Modeling of DNA local parameters predicts encrypted architectural motifs in Xenopus laevis ribosomal gene promoter.

PubMed

Roux-Rouquie, M; Marilley, M

2000-09-15

We have modeled local DNA sequence parameters to search for DNA architectural motifs involved in transcription regulation and promotion within the Xenopus laevis ribosomal gene promoter and the intergenic spacer (IGS) sequences. The IGS was found to be shaped into distinct topological domains. First, intrinsic bends split the IGS into domains of common but different helical features. Local parameters at inter-domain junctions exhibit a high variability with respect to intrinsic curvature, bendability and thermal stability. Secondly, the repeated sequence blocks of the IGS exhibit right-handed supercoiled structures which could be related to their enhancer properties. Thirdly, the gene promoter presents both inherent curvature and minor groove narrowing which may be viewed as motifs of a structural code for protein recognition and binding. Such pre-existing deformations could simply be remodeled during the binding of the transcription complex. Alternatively, these deformations could pre-shape the promoter in such a way that further remodeling is facilitated. Mutations shown to abolish promoter curvature as well as intrinsic minor groove narrowing, in a variant which maintained full transcriptional activity, bring circumstantial evidence for structurally-preorganized motifs in relation to transcription regulation and promotion. Using well documented X. laevis rDNA regulatory sequences we showed that computer modeling may be of invaluable assistance in assessing encrypted architectural motifs. The evidence of these DNA topological motifs with respect to the concept of structural code is discussed.

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

Yin, Zhiqi; Shi, Ke; Banerjee, Surajit

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNAmore » remained elusive. In this paper, we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Finally, our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.« less

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

PubMed Central

Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

2016-01-01

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

The German linguistic validation of the Ureteral Stent Symptoms Questionnaire (USSQ).

PubMed

Abt, Dominik; Dötzer, Kristina; Honek, Patrick; Müller, Karolina; Engeler, Daniel Stephan; Burger, Maximilian; Schmid, Hans-Peter; Knoll, Thomas; Sanguedolce, Francesco; Joshi, Hrishi B; Fritsche, Hans-Martin

2017-03-01

We developed and validated the German version of the Ureteral Stent Symptoms Questionnaire (USSQ) for male and female patients with indwelling ureteral stents. The German version of the USSQ was developed following a well-established multistep process. A total of 101 patients with indwelling ureteral stents completed the German USSQ as well as the validated questionnaires International Prostate Symptom Score (IPSS) or International Consultation on Incontinence Questionnaire (ICIQ) and the Short Form Health Survey (SF-36). Patients completed questionnaires at 1 and 2-4 weeks after stent insertion and 4 weeks after stent removal. Statistical analyses were performed to assess the psychometric properties of the questionnaire. The German version of the USSQ showed good internal consistency (Cronbach's α = .72-.88) and test-retest reliability [intraclass correlation coefficient (ICC) = .81-.92]. Inter-domain associations within the USSQ showed substantial correlations between different USSQ domains, indicating a high conceptual relationship of the domains. Except from urinary symptoms and general quality of life, German USSQ showed good convergent validity with the corresponding validated questionnaires. All USSQ domains showed significant sensitivity to change (p ≤ .001). The new German version of the USSQ proved to be a reliable and robust instrument for the evaluation of ureteral stent-associated morbidity for both male and female patients. It is expected to be a valid outcome measure in the future stent research.

Sequence analysis of DBL2β domain of vargene of Indonesian Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Sulistyaningsih, E.; Romadhon, B. D.; Palupi, I.; Hidayah, F.; Dewi, R.; Prasetyo, A.

2018-03-01

Malaria is a major health problem in tropical countries including Indonesia. The most deadly agent is Plasmodium falciparum. In P. falciparum infection, PfEMP1 is supposed to play an important role in the pathogenesis of malaria. PfEMP1 is encoded by var gene family, it is a polymorphic protein where the extra-cellular portion contains of three distinct binding domains: Duffy binding-like (DBL), Cysteine-rich interdomain regions (CIDR) and C2. PfEMP1 varies in domain composition and binding specificity. The study explored the characteristic of Indonesian DBL2β-var genes and investigated its role to the malaria outcome. Twenty blood samples from clinically mild to severe malaria patients in Jember, East Java were collected for DNA extraction. Diagnosis was confirmed by Giemsa-stained thick blood smear. PCR was conducted using specific primer targeting on the full-length of DBL2ß and resulted approximately single band of 1,7 kb in a sample. This band was observed only from severe malaria sample. Sequence analysis directly from PCR product showed 74-99% similarities with previous sequences in Gene Bank. In conclusion, the DBL2β domain of vargene of Indonesian isolates was 1603 nucleotides in length and there was a possible association of the existence of DBL2β domain with the severity of malaria outcome.

Horizontal gene transfer in silkworm, Bombyx mori.

PubMed

Zhu, Bo; Lou, Miao-Miao; Xie, Guan-Lin; Zhang, Guo-Qing; Zhou, Xue-Ping; Li, Bin; Jin, Gu-Lei

2011-05-19

The domesticated silkworm, Bombyx mori, is the model insect for the order Lepidoptera, has economically important values, and has gained some representative behavioral characteristics compared to its wild ancestor. The genome of B. mori has been fully sequenced while function analysis of BmChi-h and BmSuc1 genes revealed that horizontal gene transfer (HGT) maybe bestow a clear selective advantage to B. mori. However, the role of HGT in the evolutionary history of B. mori is largely unexplored. In this study, we compare the whole genome of B. mori with those of 382 prokaryotic and eukaryotic species to investigate the potential HGTs. Ten candidate HGT events were defined in B. mori by comprehensive sequence analysis using Maximum Likelihood and Bayesian method combining with EST checking. Phylogenetic analysis of the candidate HGT genes suggested that one HGT was plant-to- B. mori transfer while nine were bacteria-to- B. mori transfer. Furthermore, functional analysis based on expression, coexpression and related literature searching revealed that several HGT candidate genes have added important characters, such as resistance to pathogen, to B. mori. Results from this study clearly demonstrated that HGTs play an important role in the evolution of B. mori although the number of HGT events in B. mori is in general smaller than those of microbes and other insects. In particular, interdomain HGTs in B. mori may give rise to functional, persistent, and possibly evolutionarily significant new genes.

Stroke and aphasia quality-of-life scale-39: Reliability and validity of the Turkish version.

PubMed

Noyan-ErbaŞ, AyŞin; Toğram, Bülent

2016-10-01

The aim of this study was to adapt the stroke and aphasia quality-of-life scale-39 (SAQoL-39) to the Turkish language and carry out a reliability and validity study of the instrument in a group of patients with aphasia. The study was a descriptive study and contained three phases: adaptation of the SAQoL-39 to the Turkish language, administration of the scale to 30 aphasia patients and reliability and validity studies of the scale. Internal consistency was assessed with Cronbach's alpha and test-re-test reliability was explored (n = 14). The adaptation process was completed based on inter-rater agreement on the translated items and within the scope of final editing by the authors of the study. The SAQoL-39 in Turkish exhibited high test-re-test reliability (ICC =0.97) as well as acceptability with minimal missing data (0-1.4). This instrument exhibited high internal consistency (Cronbach's α = 0.70-0.97), domain-total correlations (r = 0.76-0.85) and inter-domain correlations (r = 0.40-0.68). The analysis shows that the Turkish version of SAQoL-39 is a scale that is highly acceptable, valid and reliable and can be easily used in evaluating the quality-of-life of Turkish people with aphasia.

Four domains: The fundamental unicell and Post-Darwinian Cognition-Based Evolution.

PubMed

Miller, William B; Torday, John S

2018-04-13

Contemporary research supports the viewpoint that self-referential cognition is the proper definition of life. From that initiating platform, a cohesive alternative evolutionary narrative distinct from standard Neodarwinism can be presented. Cognition-Based Evolution contends that biological variation is a product of a self-reinforcing information cycle that derives from self-referential attachment to biological information space-time with its attendant ambiguities. That information cycle is embodied through obligatory linkages among energy, biological information, and communication. Successive reiterations of the information cycle enact the informational architectures of the basic unicellular forms. From that base, inter-domain and cell-cell communications enable genetic and cellular variations through self-referential natural informational engineering and cellular niche construction. Holobionts are the exclusive endpoints of that self-referential cellular engineering as obligatory multicellular combinations of the essential Four Domains: Prokaryota, Archaea, Eukaryota and the Virome. Therefore, it is advocated that these Four Domains represent the perpetual object of the living circumstance rather than the visible macroorganic forms. In consequence, biology and its evolutionary development can be appraised as the continual defense of instantiated cellular self-reference. As the survival of cells is as dependent upon limitations and boundaries as upon any freedom of action, it is proposed that selection represents only one of many forms of cellular constraint that sustain self-referential integrity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design

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

Akerboom, Jasper; Velez Rivera, Jonathan D.; Rodriguez Guilbe, María M.

The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca{sup 2+}-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaMmore » and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.« less

Dynamic Communication Resource Negotiations

NASA Technical Reports Server (NTRS)

Chow, Edward; Vatan, Farrokh; Paloulian, George; Frisbie, Steve; Srostlik, Zuzana; Kalomiris, Vasilios; Apgar, Daniel

2012-01-01

Today's advanced network management systems can automate many aspects of the tactical networking operations within a military domain. However, automation of joint and coalition tactical networking across multiple domains remains challenging. Due to potentially conflicting goals and priorities, human agreement is often required before implementation into the network operations. This is further complicated by incompatible network management systems and security policies, rendering it difficult to implement automatic network management, thus requiring manual human intervention to the communication protocols used at various network routers and endpoints. This process of manual human intervention is tedious, error-prone, and slow. In order to facilitate a better solution, we are pursuing a technology which makes network management automated, reliable, and fast. Automating the negotiation of the common network communication parameters between different parties is the subject of this paper. We present the technology that enables inter-force dynamic communication resource negotiations to enable ad-hoc inter-operation in the field between force domains, without pre-planning. It also will enable a dynamic response to changing conditions within the area of operations. Our solution enables the rapid blending of intra-domain policies so that the forces involved are able to inter-operate effectively without overwhelming each other's networks with in-appropriate or un-warranted traffic. It will evaluate the policy rules and configuration data for each of the domains, then generate a compatible inter-domain policy and configuration that will update the gateway systems between the two domains.

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

PubMed Central

Guaitoli, Giambattista; Raimondi, Francesco; Gilsbach, Bernd K.; Gómez-Llorente, Yacob; Deyaert, Egon; Renzi, Fabiana; Li, Xianting; Schaffner, Adam; Jagtap, Pravin Kumar Ankush; Boldt, Karsten; von Zweydorf, Felix; Gotthardt, Katja; Lorimer, Donald D.; Yue, Zhenyu; Burgin, Alex; Janjic, Nebojsa; Sattler, Michael; Versées, Wim; Ueffing, Marius; Ubarretxena-Belandia, Iban; Kortholt, Arjan; Gloeckner, Christian Johannes

2016-01-01

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson’s disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity—together with the LRR domain—to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD. PMID:27357661

Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories

NASA Technical Reports Server (NTRS)

Nakayama, S.; Moncrief, N. D.; Kretsinger, R. H.

1992-01-01

In the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies. The main focus of this study is the classification of individual EF-hand domains. Five subfamilies--calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin--and three uniques--call, squidulin, and calcium-dependent protein kinase--are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC. The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.

Promoting interdomain analogical transfer: When creating a problem helps to solve a problem.

PubMed

Minervino, Ricardo A; Olguín, Valeria; Trench, Máximo

2017-02-01

Research on analogical thinking has devised several ways of promoting an abstract encoding of base analogs, thus rendering them more retrievable during later encounters with similar situations lacking surface similarities. Recent studies have begun to explore ways of facilitating transfer at retrieval time, which could facilitate the retrieval of distant analogs learned within contexts that were not specially directed to emphasize their abstract structure. Such studies demonstrate that comparing a target problem to an analogous problem helps students retrieve base analogs that lack surface similarities. To devise more portable ways of enhancing analogical transfer, Experiment 1 replicated Kurtz and Loewenstein's (Memory & Cognition, 35, 334-341, 2007) target-comparison procedure with an additional condition in which participants compared the target to a nonanalogous problem before attempting to reach its solution. Although comparing two analogous targets outperformed the standard transfer condition in promoting analogical transfer, comparing nonanalogous problems did not yield a transfer advantage. Based on prior studies that showed that the activity of creating analogous problems during their initial encoding elicits a more abstract representation of base analogs, in Experiment 2 we assessed whether constructing a second analogous target problem at retrieval time helps participants retrieve superficially dissimilar base analogs. As predicted, target invention increased the retrieval of distant sources. In both experiments we found an association between the quality of the generated schemas and the probability of retrieving a distant base analog from memory.

A new structural framework for integrating replication protein A into DNA processing machinery

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

Brosey, Chris; Yan, Chunli; Tsutakawa, Susan

2013-01-17

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamicmore » on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.« less

GASS-WEB: a web server for identifying enzyme active sites based on genetic algorithms.

PubMed

Moraes, João P A; Pappa, Gisele L; Pires, Douglas E V; Izidoro, Sandro C

2017-07-03

Enzyme active sites are important and conserved functional regions of proteins whose identification can be an invaluable step toward protein function prediction. Most of the existing methods for this task are based on active site similarity and present limitations including performing only exact matches on template residues, template size restraints, despite not being capable of finding inter-domain active sites. To fill this gap, we proposed GASS-WEB, a user-friendly web server that uses GASS (Genetic Active Site Search), a method based on an evolutionary algorithm to search for similar active sites in proteins. GASS-WEB can be used under two different scenarios: (i) given a protein of interest, to match a set of specific active site templates; or (ii) given an active site template, looking for it in a database of protein structures. The method has shown to be very effective on a range of experiments and was able to correctly identify >90% of the catalogued active sites from the Catalytic Site Atlas. It also managed to achieve a Matthew correlation coefficient of 0.63 using the Critical Assessment of protein Structure Prediction (CASP 10) dataset. In our analysis, GASS was ranking fourth among 18 methods. GASS-WEB is freely available at http://gass.unifei.edu.br/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Controls structures interaction, an interdisciplinary challenge for large spacecraft

NASA Technical Reports Server (NTRS)

Hanks, Brantley R.

1990-01-01

Controls structures interaction (CSI), a phenomenon which occurs when control forces interact with the flexible motion of a structure, can, if improperly treated in design and development, cause reduced performance or control instabilities. Properly applied, it can improve flexible spacecraft performance. In this paper, the NASA CSI technology program for future spacecraft applications is described. The program objectives and organization are outlined, and the nature of individual program tasks is described. The interdisciplinary aspects of CSI are also addressed.

Pareto-Zipf law in growing systems with multiplicative interactions

NASA Astrophysics Data System (ADS)

Ohtsuki, Toshiya; Tanimoto, Satoshi; Sekiyama, Makoto; Fujihara, Akihiro; Yamamoto, Hiroshi

2018-06-01

Numerical simulations of multiplicatively interacting stochastic processes with weighted selections were conducted. A feedback mechanism to control the weight w of selections was proposed. It becomes evident that when w is moderately controlled around 0, such systems spontaneously exhibit the Pareto-Zipf distribution. The simulation results are universal in the sense that microscopic details, such as parameter values and the type of control and weight, are irrelevant. The central ingredient of the Pareto-Zipf law is argued to be the mild control of interactions.

Temperature Induced Syllable Breaking Unveils Nonlinearly Interacting Timescales in Birdsong Motor Pathway

PubMed Central

Goldin, Matías A.; Alonso, Leandro M.; Alliende, Jorge A.; Goller, Franz; Mindlin, Gabriel B.

2013-01-01

The nature of telencephalic control over premotor and motor circuits is debated. Hypotheses range from complete usurping of downstream circuitry to highly interactive mechanisms of control. We show theoretically and experimentally, that telencephalic song motor control in canaries is consistent with a highly interactive strategy. As predicted from a theoretical model of respiratory control, mild cooling of a forebrain nucleus (HVC) led to song stretching, but further cooling caused progressive restructuring of song, consistent with the hypothesis that respiratory gestures are subharmonic responses to a timescale present in the output of HVC. This interaction between a life-sustaining motor function (respiration) and telencephalic song motor control suggests a more general mechanism of how nonlinear integration of evolutionarily new brain structures into existing circuitry gives rise to diverse, new behavior. PMID:23818988

Temperature induced syllable breaking unveils nonlinearly interacting timescales in birdsong motor pathway.

PubMed

Goldin, Matías A; Alonso, Leandro M; Alliende, Jorge A; Goller, Franz; Mindlin, Gabriel B

2013-01-01

The nature of telencephalic control over premotor and motor circuits is debated. Hypotheses range from complete usurping of downstream circuitry to highly interactive mechanisms of control. We show theoretically and experimentally, that telencephalic song motor control in canaries is consistent with a highly interactive strategy. As predicted from a theoretical model of respiratory control, mild cooling of a forebrain nucleus (HVC) led to song stretching, but further cooling caused progressive restructuring of song, consistent with the hypothesis that respiratory gestures are subharmonic responses to a timescale present in the output of HVC. This interaction between a life-sustaining motor function (respiration) and telencephalic song motor control suggests a more general mechanism of how nonlinear integration of evolutionarily new brain structures into existing circuitry gives rise to diverse, new behavior.

Learner Control in Hypermedia Environments

ERIC Educational Resources Information Center

Scheiter, Katharina; Gerjets, Peter

2007-01-01

Contrary to system-controlled multimedia learning environments, hypermedia systems are characterized by a high level of interactivity. This interactivity is referred to as learner control in the respective literature. For several reasons this learner control is seen as a major advantage of hypermedia for learning and instruction. For instance,…

Control and reduction of unsteady pressure loads in separated shock wave turbulent boundary layer interaction

NASA Technical Reports Server (NTRS)

Dolling, David S.; Barter, John W.

1995-01-01

The focus was on developing means of controlling and reducing unsteady pressure loads in separated shock wave turbulent boundary layer interactions. Section 1 describes how vortex generators can be used to effectively reduce loads in compression ramp interaction, while Section 2 focuses on the effects of 'boundary-layer separators' on the same interaction.

Effects of the Caregiver Interaction Profile Training on Caregiver-Child Interactions in Dutch Child Care Centers: A Randomized Controlled Trial

ERIC Educational Resources Information Center

Helmerhorst, Katrien O.; Riksen-Walraven, J. Marianne; Fukkink, Ruben G.; Tavecchio, Louis W. C.; Gevers Deynoot-Schaub, Mirjam J. J. M.

2017-01-01

Background: Previous studies underscore the need to improve caregiver-child interactions in early child care centers. Objective: In this study we used a randomized controlled trial to examine whether a 5-week video feedback training can improve six key interactive skills of caregivers in early child care centers: Sensitive responsiveness, respect…

Controlling and monitoring the space-station plasma interaction: A baseline for performing plasma experiments and using advanced technology

NASA Technical Reports Server (NTRS)

Whipple, Elden C.; Olsen, Richard C.

1986-01-01

The size, complexity, and motion of space station through the Earth's environmental plasma means that there will be a large, complicated interaction region, involving a sheath, wake, charging of surfaces, induced electric fields, secondary emission, outgassing with ionization, etc. This interaction will necessarily be a factor in carrying out and interpreting plasma experiments and in the use of certain technologies. Attention should be given ahead of time to: (1) monitoring this interaction so that it is well described; (2) implifying the interaction by appropriate design and construction of the spacecraft and by appropriate planning of technology use; and (3) controlling the interaction by both active and passive means. Plasma emitters for modifying and controlling the spacecraft charge should be placed in several locations. Portable electrostatic shields could be deployed around noisy sections of the spacecraft in order to carry out sensitive experiments. A particle umbrella could be raised to deflect the ram ions and neutrals in order to provide a controlled environment. These interactions are briefly discussed.

Measuring interactivity on tobacco control websites.

PubMed

Freeman, Becky; Chapman, Simon

2012-08-01

With the increased reach of Web 2.0, Internet users expect webpages to be interactive. No studies have been conducted to assess whether tobacco control-relevant sites have implemented these features. The authors conducted an analysis of an international sample of tobacco control-relevant websites to determine their level of interactivity. The sample included 68 unique websites selected from Google searches in 5 countries, on each country's Google site, using the term smoking. The 68 sites were analyzed for 10 categories of interactive tools. The most common type of interactive content found on 46 (68%) of sites was for multimedia featuring content that was not primarily text based, such as photo galleries, videos, or podcasts. Only 11 (16%) websites-outside of media sites-allowed people to interact and engage with the site owners and other users by allowing posting comments on content and/or hosting forums/discussions. Linkages to social networking sites were low: 17 pages (25%) linked to Twitter, 15 (22%) to Facebook, and 11 (16%) to YouTube. Interactivity and connectedness to online social media appears to still be in its infancy among tobacco control-relevant sites.

User Manual for Beta Version of TURBO-GRD: A Software System for Interactive Two-Dimensional Boundary/ Field Grid Generation, Modification, and Refinement

NASA Technical Reports Server (NTRS)

Choo, Yung K.; Slater, John W.; Henderson, Todd L.; Bidwell, Colin S.; Braun, Donald C.; Chung, Joongkee

1998-01-01

TURBO-GRD is a software system for interactive two-dimensional boundary/field grid generation. modification, and refinement. Its features allow users to explicitly control grid quality locally and globally. The grid control can be achieved interactively by using control points that the user picks and moves on the workstation monitor or by direct stretching and refining. The techniques used in the code are the control point form of algebraic grid generation, a damped cubic spline for edge meshing and parametric mapping between physical and computational domains. It also performs elliptic grid smoothing and free-form boundary control for boundary geometry manipulation. Internal block boundaries are constructed and shaped by using Bezier curve. Because TURBO-GRD is a highly interactive code, users can read in an initial solution, display its solution contour in the background of the grid and control net, and exercise grid modification using the solution contour as a guide. This process can be called an interactive solution-adaptive grid generation.

Controls-Structures Interaction (CSI) technology program summary. Earth orbiting platforms program area of the space platforms technology program

NASA Technical Reports Server (NTRS)

Newsom, Jerry R.

1991-01-01

Control-Structures Interaction (CSI) technology embraces the understanding of the interaction between the spacecraft structure and the control system, and the creation and validation of concepts, techniques, and tools, for enabling the interdisciplinary design of an integrated structure and control system, rather than the integration of a structural design and a control system design. The goal of this program is to develop validated CSI technology for integrated design/analysis and qualification of large flexible space systems and precision space structures. A description of the CSI technology program is presented.

Learner Control versus Program Control in Interactive Videodisc Instruction: What Are the Effects in Procedural Learning?

ERIC Educational Resources Information Center

Shyu, Hsin-Yih; Brown, Scott W.

1992-01-01

Discussion of learner-controlled instruction focuses on a study of undergraduates that compared learner control with program control in interactive videodisc instruction for making an origami crane. Student performance, self-efficacy concerning the instructional task, time on task, and student attitudes toward the instruction are examined. (32…

Analysis of airframe/engine interactions in integrated flight and propulsion control

NASA Technical Reports Server (NTRS)

Schierman, John D.; Schmidt, David K.

1991-01-01

An analysis framework for the assessment of dynamic cross-coupling between airframe and engine systems from the perspective of integrated flight/propulsion control is presented. This analysis involves to determining the significance of the interactions with respect to deterioration in stability robustness and performance, as well as critical frequency ranges where problems may occur due to these interactions. The analysis illustrated here investigates both the airframe's effects on the engine control loops and the engine's effects on the airframe control loops in two case studies. The second case study involves a multi-input/multi-output analysis of the airframe. Sensitivity studies are performed on critical interactions to examine the degradations in the system's stability robustness and performance. Magnitudes of the interactions required to cause instabilities, as well as the frequencies at which the instabilities occur are recorded. Finally, the analysis framework is expanded to include control laws which contain cross-feeds between the airframe and engine systems.

Simple F Test Reveals Gene-Gene Interactions in Case-Control Studies

PubMed Central

Chen, Guanjie; Yuan, Ao; Zhou, Jie; Bentley, Amy R.; Adeyemo, Adebowale; Rotimi, Charles N.

2012-01-01

Missing heritability is still a challenge for Genome Wide Association Studies (GWAS). Gene-gene interactions may partially explain this residual genetic influence and contribute broadly to complex disease. To analyze the gene-gene interactions in case-control studies of complex disease, we propose a simple, non-parametric method that utilizes the F-statistic. This approach consists of three steps. First, we examine the joint distribution of a pair of SNPs in cases and controls separately. Second, an F-test is used to evaluate the ratio of dependence in cases to that of controls. Finally, results are adjusted for multiple tests. This method was used to evaluate gene-gene interactions that are associated with risk of Type 2 Diabetes among African Americans in the Howard University Family Study. We identified 18 gene-gene interactions (P < 0.0001). Compared with the commonly-used logistical regression method, we demonstrate that the F-ratio test is an efficient approach to measuring gene-gene interactions, especially for studies with limited sample size. PMID:22837643

76 FR 72215 - Certain Products Containing Interactive Program Guide and Parental Controls Technology; Receipt...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-22

... Parental Controls Technology; Receipt of Complaint; Solicitation of Comments Relating to the Public... Containing Interactive Program Guide and Parental Controls Technology, DN 2856; the Commission is soliciting... parental controls technology. The complaint names Vizio, Inc. of Irvine, CA; Haier Group Corp. of China...

An Affect Control Theory of Technology

ERIC Educational Resources Information Center

Shank, Daniel B.

2010-01-01

Affect control theory is a theory of interaction that takes into account cultural meanings. Affect control research has previously considered interaction with technology, but there remains a lack of theorizing about inclusion of technology within the theory. This paper lays a foundation for an affect control theory of technology by addressing key…

Examining the interaction between cognitive control and reward sensitivity in substance use dependence.

PubMed

Charles-Walsh, Kathleen; Upton, Daniel J; Hester, Robert

2016-09-01

Drug dependence is characterized by altered reward processing and poor cognitive control, expressed as a preference for immediate rewards and impaired inhibitory control, respectively. To examine the interaction between reward processing (via the presence or absence of reward) and mechanisms of inhibitory control in drug dependence, the current study used the Monetary Incentive Control Task (MICT) to examine whether a group of opiate dependent persons demonstrated greater difficulty exerting control over immediate rewards compared to neutral stimuli. The MICT is a Go/Stop paradigm that examines inhibitory control over immediate rewards. Performance of 32 opiate dependent individuals was compared to 29 healthy controls. Opiate users demonstrated poorer inhibitory performance than controls, irrespective of cues signaling immediate reward. Whereas control participants' responses were modulated by probability cues, the opiate group did not show a capacity to up-regulate their cognitive control performance. The present results suggest a general decrease in cognitive control in opiate dependence, accompanied by a reduced ability to optimally modulate behavior in accordance with external cues. Opiate users and controls did not differ in the interaction between cognitive control and reward. The study highlights important issues for future research to consider when further examining this interaction in drug dependence. Crown Copyright © 2016. Published by Elsevier Ireland Ltd. All rights reserved.

Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family

PubMed Central

Uhart, Marina; Flores, Gabriel; Bustos, Diego M.

2016-01-01

Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

Task-dependent and distinct roles of the temporoparietal junction and inferior frontal cortex in the control of imitation

PubMed Central

Obhi, Sukhvinder S.; Banissy, Michael J.; Santiesteban, Idalmis; Press, Clare; Catmur, Caroline; Bird, Geoffrey

2015-01-01

The control of neurological networks supporting social cognition is crucially important for social interaction. In particular, the control of imitation is directly linked to interaction quality, with impairments associated with disorders characterized by social difficulties. Previous work suggests inferior frontal cortex (IFC) and the temporoparietal junction (TPJ) are involved in controlling imitation, but the functional roles of these areas remain unclear. Here, transcranial direct current stimulation (tDCS) was used to enhance cortical excitability at IFC and the TPJ prior to the completion of three tasks: (i) a naturalistic social interaction during which increased imitation is known to improve rapport, (ii) a choice reaction time task in which imitation needs to be inhibited for successful performance and (iii) a non-imitative control task. Relative to sham stimulation, stimulating IFC improved the context-dependent control of imitation—participants imitated more during the social interaction and less during the imitation inhibition task. In contrast, stimulating the TPJ reduced imitation in the inhibition task without affecting imitation during social interaction. Neither stimulation site affected the non-imitative control task. These data support a model in which IFC modulates imitation directly according to task demands, whereas TPJ controls task-appropriate shifts in attention toward representation of the self or the other, indirectly impacting upon imitation. PMID:25481003

Performance-Driven Hybrid Full-Body Character Control for Navigation and Interaction in Virtual Environments

NASA Astrophysics Data System (ADS)

Mousas, Christos; Anagnostopoulos, Christos-Nikolaos

2017-06-01

This paper presents a hybrid character control interface that provides the ability to synthesize in real-time a variety of actions based on the user's performance capture. The proposed methodology enables three different performance interaction modules: the performance animation control that enables the direct mapping of the user's pose to the character, the motion controller that synthesizes the desired motion of the character based on an activity recognition methodology, and the hybrid control that lies within the performance animation and the motion controller. With the methodology presented, the user will have the freedom to interact within the virtual environment, as well as the ability to manipulate the character and to synthesize a variety of actions that cannot be performed directly by him/her, but which the system synthesizes. Therefore, the user is able to interact with the virtual environment in a more sophisticated fashion. This paper presents examples of different scenarios based on the three different full-body character control methodologies.

Virus-Like Particle Secretion and Genotype-Dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine

PubMed Central

Galula, Jedhan U.; Shen, Wen-Fan; Chuang, Shih-Te

2014-01-01

ABSTRACT Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. IMPORTANCE Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine candidates were evaluated for their immunogenicity, homologous and heterologous neutralizing (Nt) antibody titers, and cross-genotype protection in a murine model. The immunity elicited by our prototype vaccine candidate (Asian 1 genotype strain 16681) in mice was protective against viruses of other genotypes but not against virus of the Sylvatic genotype, whose emergence and potential risk after introduction into the human population have previously been demonstrated. The underlying mechanism of a lack of protection elicited by the prototype vaccine may at least be contributed by the absence of a flavivirus subgroup-cross-reactive, highly neutralizing monoclonal antibody 1B7-5-like epitope in DENV-2 of the Sylvatic genotype. The DENV DNA vaccine directs the synthesis and assembly of virus-like particles (VLPs) and induces immune responses similar to those elicited by live-attenuated vaccines, and its flexibility permits the fast deployment of vaccine to combat emerging viruses, such as Sylvatic genotype viruses. The enhanced VLP secretion obtained by replacement of ectodomain I-II (EDI-II) of the Cosmopolitan genotype vaccine construct (VD2-Cosmopolitan) with the Asian 1 EDI-II elicited significantly higher total IgG and Nt antibody titers and suggests a novel approach to enhance the immunogenicity of the DNA vaccine. A DENV vaccine capable of eliciting protective immunity against viruses of existing and emerging genotypes should be the focus of future DENV vaccine development. PMID:25008922

Control of a Robot Dancer for Enhancing Haptic Human-Robot Interaction in Waltz.

PubMed

Hongbo Wang; Kosuge, K

2012-01-01

Haptic interaction between a human leader and a robot follower in waltz is studied in this paper. An inverted pendulum model is used to approximate the human's body dynamics. With the feedbacks from the force sensor and laser range finders, the robot is able to estimate the human leader's state by using an extended Kalman filter (EKF). To reduce interaction force, two robot controllers, namely, admittance with virtual force controller, and inverted pendulum controller, are proposed and evaluated in experiments. The former controller failed the experiment; reasons for the failure are explained. At the same time, the use of the latter controller is validated by experiment results.

Parametric excitation and squeezing in a many-body spinor condensate

PubMed Central

Hoang, T. M.; Anquez, M.; Robbins, B. A.; Yang, X. Y.; Land, B. J.; Hamley, C. D.; Chapman, M. S.

2016-01-01

Atomic spins are usually manipulated using radio frequency or microwave fields to excite Rabi oscillations between different spin states. These are single-particle quantum control techniques that perform ideally with individual particles or non-interacting ensembles. In many-body systems, inter-particle interactions are unavoidable; however, interactions can be used to realize new control schemes unique to interacting systems. Here we demonstrate a many-body control scheme to coherently excite and control the quantum spin states of an atomic Bose gas that realizes parametric excitation of many-body collective spin states by time varying the relative strength of the Zeeman and spin-dependent collisional interaction energies at multiples of the natural frequency of the system. Although parametric excitation of a classical system is ineffective from the ground state, we show that in our experiment, parametric excitation from the quantum ground state leads to the generation of quantum squeezed states. PMID:27044675

Analysis of airframe/engine interactions - An integrated control perspective

NASA Technical Reports Server (NTRS)

Schmidt, David K.; Schierman, John D.; Garg, Sanjay

1990-01-01

Techniques for the analysis of the dynamic interactions between airframe/engine dynamical systems are presented. Critical coupling terms are developed that determine the significance of these interactions with regard to the closed loop stability and performance of the feedback systems. A conceptual model is first used to indicate the potential sources of the coupling, how the coupling manifests itself, and how the magnitudes of these critical coupling terms are used to quantify the effects of the airframe/engine interactions. A case study is also presented involving an unstable airframe with thrust vectoring for attitude control. It is shown for this system with classical, decentralized control laws that there is little airframe/engine interaction, and the stability and performance with those control laws is not affected. Implications of parameter uncertainty in the coupling dynamics is also discussed, and effects of these parameter variations are also demonstrated to be small for this vehicle configuration.

Parametric excitation and squeezing in a many-body spinor condensate

NASA Astrophysics Data System (ADS)

Hoang, T. M.; Anquez, M.; Robbins, B. A.; Yang, X. Y.; Land, B. J.; Hamley, C. D.; Chapman, M. S.

2016-04-01

Atomic spins are usually manipulated using radio frequency or microwave fields to excite Rabi oscillations between different spin states. These are single-particle quantum control techniques that perform ideally with individual particles or non-interacting ensembles. In many-body systems, inter-particle interactions are unavoidable; however, interactions can be used to realize new control schemes unique to interacting systems. Here we demonstrate a many-body control scheme to coherently excite and control the quantum spin states of an atomic Bose gas that realizes parametric excitation of many-body collective spin states by time varying the relative strength of the Zeeman and spin-dependent collisional interaction energies at multiples of the natural frequency of the system. Although parametric excitation of a classical system is ineffective from the ground state, we show that in our experiment, parametric excitation from the quantum ground state leads to the generation of quantum squeezed states.

Epidemic spreading on random surfer networks with optimal interaction radius

NASA Astrophysics Data System (ADS)

Feng, Yun; Ding, Li; Hu, Ping

2018-03-01

In this paper, the optimal control problem of epidemic spreading on random surfer heterogeneous networks is considered. An epidemic spreading model is established according to the classification of individual's initial interaction radii. Then, a control strategy is proposed based on adjusting individual's interaction radii. The global stability of the disease free and endemic equilibrium of the model is investigated. We prove that an optimal solution exists for the optimal control problem and the explicit form of which is presented. Numerical simulations are conducted to verify the correctness of the theoretical results. It is proved that the optimal control strategy is effective to minimize the density of infected individuals and the cost associated with the adjustment of interaction radii.

NASA/DOD Control/Structures Interaction Technology, 1986

NASA Technical Reports Server (NTRS)

Wright, Robert L. (Compiler)

1986-01-01

Control/structures interactions, deployment dynamics and system performance of large flexible spacecraft are discussed. Spacecraft active controls, deployable truss structures, deployable antennas, solar power systems for space stations, pointing control systems for space station gimballed payloads, computer-aided design for large space structures, and passive damping for flexible structures are among the topics covered.

Active Voodoo Dolls: A Vision Based Input Device for Nonrigid Control.

DTIC Science & Technology

1998-08-01

A vision based technique for nonrigid control is presented that can be used for animation and video game applications. The user grasps a soft...allowing the user to control it interactively. Our use of texture mapping hardware in tracking makes the system responsive enough for interactive animation and video game character control.

Subliminally and consciously induced cognitive conflicts interact at several processing levels.

PubMed

Stock, Ann-Kathrin; Friedrich, Julia; Beste, Christian

2016-12-01

Controlled behavior is susceptible to conflicts that can emerge from subliminal or consciously processed information. While research suggests that both sources of conflicting information may interact in their modulation of controlled behavior, it has remained unclear which cognitive sub-processes involved in controlled behavior are affected by this interaction; i.e., at which processing level subliminally and consciously induced response conflicts interact in modulating controlled behavior. Moreover, we investigated whether this interaction of subliminally and consciously induced response conflicts was due to a nexus between the two types of conflict like a common cognitive process or factor. For this, n = 38 healthy young subjects completed a paradigm which combines subliminal primes and consciously perceived flankers while an electroencephalography (EEG) was recorded. We show that the interaction of subliminal and conscious sources of conflict is not restricted to the response selection level (N2) but can already be shown at the earliest stages of perceptual and attentional processing (P1). While the degree of early attentional processing of subliminal information seems to depend on the absence of consciously perceived response conflicts, conflicts during the stage of response selection may be either reduced or enhanced by subliminal priming. Moreover, the results showed that even though the two different sources of conflict interact at the response selection level, they clearly originate from two distinct processes that interact before they detrimentally affect cognitive control. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Formal Investigation of Human Spatial Control Skills: Mathematical Formalization, Skill Development, and Skill Assessment

NASA Astrophysics Data System (ADS)

Li, Bin

Spatial control behaviors account for a large proportion of human everyday activities from normal daily tasks, such as reaching for objects, to specialized tasks, such as driving, surgery, or operating equipment. These behaviors involve intensive interactions within internal processes (i.e. cognitive, perceptual, and motor control) and with the physical world. This dissertation builds on a concept of interaction pattern and a hierarchical functional model. Interaction pattern represents a type of behavior synergy that humans coordinates cognitive, perceptual, and motor control processes. It contributes to the construction of the hierarchical functional model that delineates humans spatial control behaviors as the coordination of three functional subsystems: planning, guidance, and tracking/pursuit. This dissertation formalizes and validates these two theories and extends them for the investigation of human spatial control skills encompassing development and assessment. Specifically, this dissertation first presents an overview of studies in human spatial control skills encompassing definition, characteristic, development, and assessment, to provide theoretical evidence for the concept of interaction pattern and the hierarchical functional model. The following, the human experiments for collecting motion and gaze data and techniques to register and classify gaze data, are described. This dissertation then elaborates and mathematically formalizes the hierarchical functional model and the concept of interaction pattern. These theories then enables the construction of a succinct simulation model that can reproduce a variety of human performance with a minimal set of hypotheses. This validates the hierarchical functional model as a normative framework for interpreting human spatial control behaviors. The dissertation then investigates human skill development and captures the emergence of interaction pattern. The final part of the dissertation applies the hierarchical functional model for skill assessment and introduces techniques to capture interaction patterns both from the top down using their geometric features and from the bottom up using their dynamical characteristics. The validity and generality of the skill assessment is illustrated using two the remote-control flight and laparoscopic surgical training experiments.

Effect of cutoff radius, long range interaction and temperature controller on thermodynamic properties of fluids: Methanol as an example

NASA Astrophysics Data System (ADS)

Obeidat, Abdalla; Jaradat, Adnan; Hamdan, Bushra; Abu-Ghazleh, Hind

2018-04-01

The best spherical cutoff radius, long range interaction and temperature controller were determined using surface tension, density, and diffusion coefficients of van Leeuwen and Smit methanol. A quite good range of cutoff radii from 0.75 to 1.45 nm has been studied on Coulomb cut-off and particle mesh Ewald (PME) long range interaction to determine the best cutoff radius and best long range interaction as well for four sets of temperature: 200, 230, 270 and 300 K. To determine the best temperature controller, the cutoff radius of 1.25 nm was fixed using PME long range interaction on calculating the above properties at low temperature range: 200-300 K.

Orbiter subsystem hardware/software interaction analysis. Volume 8: Forward reaction control system

NASA Technical Reports Server (NTRS)

Becker, D. D.

1980-01-01

The results of the orbiter hardware/software interaction analysis for the AFT reaction control system are presented. The interaction between hardware failure modes and software are examined in order to identify associated issues and risks. All orbiter subsystems and interfacing program elements which interact with the orbiter computer flight software are analyzed. The failure modes identified in the subsystem/element failure mode and effects analysis are discussed.

Collaborative Micro Aerial Vehicle Exploration of Outdoor Environments

DTIC Science & Technology

2010-02-01

with accelerometer or multitouch capabilities have also simply followed the same WYSIWYG paradigm. Finally, no HRI research exists on interacting ...Nudge Control relies on multimodal interaction ( multitouch gestures and tilting) to create a rich control interaction without cluttering the display...used in one of two different modes. NG mode uses a set of tilting gestures while CT mode uses multitouch gestures to interact with the vehicle. Both of

The physicochemical process of bacterial attachment to abiotic surfaces: Challenges for mechanistic studies, predictability and the development of control strategies.

PubMed

Wang, Yi; Lee, Sui Mae; Dykes, Gary

2015-01-01

Bacterial attachment to abiotic surfaces can be explained as a physicochemical process. Mechanisms of the process have been widely studied but are not yet well understood due to their complexity. Physicochemical processes can be influenced by various interactions and factors in attachment systems, including, but not limited to, hydrophobic interactions, electrostatic interactions and substratum surface roughness. Mechanistic models and control strategies for bacterial attachment to abiotic surfaces have been established based on the current understanding of the attachment process and the interactions involved. Due to a lack of process control and standardization in the methodologies used to study the mechanisms of bacterial attachment, however, various challenges are apparent in the development of models and control strategies. In this review, the physicochemical mechanisms, interactions and factors affecting the process of bacterial attachment to abiotic surfaces are described. Mechanistic models established based on these parameters are discussed in terms of their limitations. Currently employed methods to study these parameters and bacterial attachment are critically compared. The roles of these parameters in the development of control strategies for bacterial attachment are reviewed, and the challenges that arise in developing mechanistic models and control strategies are assessed.

Preliminary control/structure interaction study of coupled Space Station Freedom/Assembly Work Platform/orbiter

NASA Technical Reports Server (NTRS)

Singh, Sudeep K.; Lindenmoyer, Alan J.

1989-01-01

Results are presented from a preliminary control/structure interaction study of the Space Station, the Assembly Work Platform, and the STS orbiter dynamics coupled with the orbiter and station control systems. The first three Space Station assembly flight configurations and their finite element representations are illustrated. These configurations are compared in terms of control authority in each axis and propellant usage. The control systems design parameters during assembly are computed. Although the rigid body response was acceptable with the orbiter Primary Reaction Control System, the flexible body response showed large structural deflections and loads. It was found that severe control/structure interaction occurred if the stiffness of the Assembly Work Platform was equal to that of the station truss. Also, the response of the orbiter Vernier Reaction Control System to small changes in inertia properties is examined.

Controlling Second Harmonic Efficiency of Laser Beam Interactions

NASA Technical Reports Server (NTRS)

Barnes, Norman P. (Inventor); Walsh, Brian M. (Inventor); Reichle, Donald J. (Inventor)

2011-01-01

A method is provided for controlling second harmonic efficiency of laser beam interactions. A laser system generates two laser beams (e.g., a laser beam with two polarizations) for incidence on a nonlinear crystal having a preferred direction of propagation. Prior to incidence on the crystal, the beams are optically processed based on the crystal's beam separation characteristics to thereby control a position in the crystal along the preferred direction of propagation at which the beams interact.

Control strategies for robots in contact

NASA Astrophysics Data System (ADS)

Park, Jaeheung

In the field of robotics, there is a growing need to provide robots with the ability to interact with complex and unstructured environments. Operations in such environments pose significant challenges in terms of sensing, planning, and control. In particular, it is critical to design control algorithms that account for the dynamics of the robot and environment at multiple contacts. The work in this thesis focuses on the development of a control framework that addresses these issues. The approaches are based on the operational space control framework and estimation methods. By accounting for the dynamics of the robot and environment, modular and systematic methods are developed for robots interacting with the environment at multiple locations. The proposed force control approach demonstrates high performance in the presence of uncertainties. Building on this basic capability, new control algorithms have been developed for haptic teleoperation, multi-contact interaction with the environment, and whole body motion of non-fixed based robots. These control strategies have been experimentally validated through simulations and implementations on physical robots. The results demonstrate the effectiveness of the new control structure and its robustness to uncertainties. The contact control strategies presented in this thesis are expected to contribute to the needs in advanced controller design for humanoid and other complex robots interacting with their environments.

Effects of the Caregiver Interaction Profile Training on Caregiver-Child Interactions in Dutch Child Care Centers: A Randomized Controlled Trial.

PubMed

Helmerhorst, Katrien O W; Riksen-Walraven, J Marianne A; Fukkink, Ruben G; Tavecchio, Louis W C; Gevers Deynoot-Schaub, Mirjam J J M

2017-01-01

Previous studies underscore the need to improve caregiver-child interactions in early child care centers. In this study we used a randomized controlled trial to examine whether a 5-week video feedback training can improve six key interactive skills of caregivers in early child care centers: Sensitive responsiveness, respect for autonomy, structuring and limit setting, verbal communication, developmental stimulation, and fostering positive peer interactions. A total of 139 caregivers from 68 early child care groups for 0- to 4-year-old children in Dutch child care centers participated in this RCT, 69 in the intervention condition and 70 in the control condition. Caregiver interactive skills during everyday interactions with the children were rated from videotape using the Caregiver Interaction Profile (CIP) scales at pretest, posttest, and follow-up 3 months after the posttest. Results at posttest indicate a significant positive training effect on all six caregiver interactive skills. Effect sizes of the CIP training range between d  = 0.35 and d  = 0.79. Three months after the posttest, caregivers in the intervention group still scored significantly higher on sensitive responsiveness, respect for autonomy, verbal communication, and fostering positive peer interactions than caregivers in the control group with effect sizes ranging between d  = 0.47 and d  = 0.70. This study shows that the quality of caregiver-child interactions can be improved for all six important caregiver skills, with a relatively short training program. Possible ways to further improve the training and to implement it in practice and education are discussed.

Task-dependent and distinct roles of the temporoparietal junction and inferior frontal cortex in the control of imitation.

PubMed

Hogeveen, Jeremy; Obhi, Sukhvinder S; Banissy, Michael J; Santiesteban, Idalmis; Press, Clare; Catmur, Caroline; Bird, Geoffrey

2015-07-01

The control of neurological networks supporting social cognition is crucially important for social interaction. In particular, the control of imitation is directly linked to interaction quality, with impairments associated with disorders characterized by social difficulties. Previous work suggests inferior frontal cortex (IFC) and the temporoparietal junction (TPJ) are involved in controlling imitation, but the functional roles of these areas remain unclear. Here, transcranial direct current stimulation (tDCS) was used to enhance cortical excitability at IFC and the TPJ prior to the completion of three tasks: (i) a naturalistic social interaction during which increased imitation is known to improve rapport, (ii) a choice reaction time task in which imitation needs to be inhibited for successful performance and (iii) a non-imitative control task. Relative to sham stimulation, stimulating IFC improved the context-dependent control of imitation-participants imitated more during the social interaction and less during the imitation inhibition task. In contrast, stimulating the TPJ reduced imitation in the inhibition task without affecting imitation during social interaction. Neither stimulation site affected the non-imitative control task. These data support a model in which IFC modulates imitation directly according to task demands, whereas TPJ controls task-appropriate shifts in attention toward representation of the self or the other, indirectly impacting upon imitation. © The Author (2014). Published by Oxford University Press.

Interactive Metronome training in children with attention deficit and developmental coordination disorders.

PubMed

Cosper, Sharon M; Lee, Gregory P; Peters, Susan Beth; Bishop, Elizabeth

2009-12-01

The objective of this study was to examine the efficacy of Interactive Metronome (Interactive Metronome, Sunrise, Florida, USA) training in a group of children with mixed attentional and motor coordination disorders to further explore which subcomponents of attentional control and motor functioning the training influences. Twelve children who had been diagnosed with attention deficit hyperactivity disorder, in conjunction with either developmental coordination disorder (n=10) or pervasive developmental disorder (n=2), underwent 15 1-h sessions of Interactive Metronome training over a 15-week period. Each child was assessed before and after the treatment using measures of attention, coordination, and motor control to determine the efficacy of training on these cognitive and behavioral realms. As a group, the children made significant improvements in complex visual choice reaction time and visuomotor control after the training. There were, however, no significant changes in sustained attention or inhibitory control over inappropriate motor responses after treatment. These results suggest Interactive Metronome training may address deficits in visuomotor control and speed, but appears to have little effect on sustained attention or motor inhibition.

Prosthetic Leg Control in the Nullspace of Human Interaction.

PubMed

Gregg, Robert D; Martin, Anne E

2016-07-01

Recent work has extended the control method of virtual constraints, originally developed for autonomous walking robots, to powered prosthetic legs for lower-limb amputees. Virtual constraints define desired joint patterns as functions of a mechanical phasing variable, which are typically enforced by torque control laws that linearize the output dynamics associated with the virtual constraints. However, the output dynamics of a powered prosthetic leg generally depend on the human interaction forces, which must be measured and canceled by the feedback linearizing control law. This feedback requires expensive multi-axis load cells, and actively canceling the interaction forces may minimize the human's influence over the prosthesis. To address these limitations, this paper proposes a method for projecting virtual constraints into the nullspace of the human interaction terms in the output dynamics. The projected virtual constraints naturally render the output dynamics invariant with respect to the human interaction forces, which instead enter into the internal dynamics of the partially linearized prosthetic system. This method is illustrated with simulations of a transfemoral amputee model walking with a powered knee-ankle prosthesis that is controlled via virtual constraints with and without the proposed projection.

Aggregation state and magnetic properties of magnetite nanoparticles controlled by an optimized silica coating

NASA Astrophysics Data System (ADS)

Pérez, Nicolás; Moya, C.; Tartaj, P.; Labarta, A.; Batlle, X.

2017-01-01

The control of magnetic interactions is becoming essential to expand/improve the applicability of magnetic nanoparticles (NPs). Here, we show that an optimized microemulsion method can be used to obtain homogenous silica coatings on even single magnetic nuclei of highly crystalline Fe3-xO4 NPs (7 and 16 nm) derived from a high-temperature method. We show that the thickness of this coating is controlled almost at will allowing much higher average separation among particles as compared to the oleic acid coating present on pristine NPs. Magnetic susceptibility studies show that the thickness of the silica coating allows the control of magnetic interactions. Specifically, as this effect is better displayed for the smallest particles, we show that dipole-dipole interparticle interactions can be tuned progressively for the 7 nm NPs, from almost non-interacting to strongly interacting particles at room temperature. The quantitative analysis of the magnetic properties unambiguously suggests that dipolar interactions significantly broaden the effective distribution of energy barriers by spreading the distribution of activation magnetic volumes.

Latent interaction effects in the theory of planned behaviour applied to quitting smoking.

PubMed

Hukkelberg, Silje Sommer; Hagtvet, Knut A; Kovac, Velibor Bobo

2014-02-01

This study applies three latent interaction models in the theory of planned behaviour (TPB; Ajzen, 1988, Attitudes, personality, and behavior. Homewood, IL: Dorsey Press; Ajzen, 1991, Organ. Behav. Hum. Decis. Process., 50, 179) to quitting smoking: (1) attitude × perceived behavioural control on intention; (2) subjective norms (SN) × attitude on intention; and (3) perceived behavioural control × intention on quitting behaviour. The data derive from a longitudinal Internet survey of 939 smokers aged 15-74 over a period of 4 months. Latent interaction effects were estimated using the double-mean-centred unconstrained approach (Lin et al., 2010, Struct. Equ. Modeling, 17, 374) in LISREL. Attitude × SN and attitude × perceived behavioural control both showed a significant interaction effect on intention. No significant interaction effect was found for perceived behavioural control × intention on quitting. The latent interaction approach is a useful method for investigating specific conditions between TPB components in the context of quitting behaviour. Theoretical and practical implications of the results are discussed. © 2013 The British Psychological Society.

NASA/DOD Control/Structures Interaction Technology, 1986

NASA Technical Reports Server (NTRS)

Wright, Robert L. (Compiler)

1987-01-01

Papers presented at the CSI Technology Conference are given. The conference was jointly sponsored by the NASA Office of Aeronautics and Space Technology and the Department of Defense. The conference is the beginning of a series of annual conferences whose purpose is to report to industry, academia, and government agencies the current status of Control/Structures Interaction technology. The conference program was divided into five sessions: (1) Future spacecraft requirements; Technology issues and impact; (2) DOD special topics; (3) Large space systems technology; (4) Control of flexible structures, and (5) Selected NASA research in control structures interaction.

Representing and Learning Complex Object Interactions

PubMed Central

Zhou, Yilun; Konidaris, George

2017-01-01

We present a framework for representing scenarios with complex object interactions, in which a robot cannot directly interact with the object it wishes to control, but must instead do so via intermediate objects. For example, a robot learning to drive a car can only indirectly change its pose, by rotating the steering wheel. We formalize such complex interactions as chains of Markov decision processes and show how they can be learned and used for control. We describe two systems in which a robot uses learning from demonstration to achieve indirect control: playing a computer game, and using a hot water dispenser to heat a cup of water. PMID:28593181

Influence of air-jet vortex generator diameter on separation region

NASA Astrophysics Data System (ADS)

Szwaba, Ryszard

2013-08-01

Control of shock wave and boundary layer interaction continues to attract a lot of attention. In recent decades several methods of interaction control have been investigated. The research has mostly concerned solid (vane type) vortex generators and transpiration methods of suction and blowing. This investigation concerns interaction control using air-jets to generate streamwise vortices. The effectiveness of air-jet vortex generators in controlling separation has been proved in a previous research. The present paper focuses on the influence of the vortex generator diameter on the separation region. It presents the results of experimental investigations and provides new guidelines for the design of air-jet vortex generators to obtain more effective separation control.

Model-Based Design of Air Traffic Controller-Automation Interaction

NASA Technical Reports Server (NTRS)

Romahn, Stephan; Callantine, Todd J.; Palmer, Everett A.; Null, Cynthia H. (Technical Monitor)

1998-01-01

A model of controller and automation activities was used to design the controller-automation interactions necessary to implement a new terminal area air traffic management concept. The model was then used to design a controller interface that provides the requisite information and functionality. Using data from a preliminary study, the Crew Activity Tracking System (CATS) was used to help validate the model as a computational tool for describing controller performance.

A large number of stepping motor network construction by PLC

NASA Astrophysics Data System (ADS)

Mei, Lin; Zhang, Kai; Hongqiang, Guo

2017-11-01

In the flexible automatic line, the equipment is complex, the control mode is flexible, how to realize the large number of step and servo motor information interaction, the orderly control become a difficult control. Based on the existing flexible production line, this paper makes a comparative study of its network strategy. After research, an Ethernet + PROFIBUSE communication configuration based on PROFINET IO and profibus was proposed, which can effectively improve the data interaction efficiency of the equipment and stable data interaction information.

SIG -- The Role of Human-Computer Interaction in Next-Generation Control Rooms

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

Ronald L. Boring; Jacques Hugo; Christian Richard

2005-04-01

The purpose of this CHI Special Interest Group (SIG) is to facilitate the convergence between human-computer interaction (HCI) and control room design. HCI researchers and practitioners actively need to infuse state-of-the-art interface technology into control rooms to meet usability, safety, and regulatory requirements. This SIG outlines potential HCI contributions to instrumentation and control (I&C) and automation in control rooms as well as to general control room design.

Effects of the Orion Launch Abort Vehicle Plumes on Aerodynamics and Controllability

NASA Technical Reports Server (NTRS)

Vicker, Darby; Childs, Robert; Rogers,Stuart E.; McMullen, Matthew; Garcia, Joseph; Greathouse, James

2013-01-01

Characterization of the launch abort system of the Multi-purpose Crew Vehicle (MPCV) for control design and accurate simulation has provided a significant challenge to aerodynamicists and design engineers. The design space of the launch abort vehicle (LAV) includes operational altitudes from ground level to approximately 300,000 feet, Mach numbers from 0-9, and peak dynamic pressure near 1300psf during transonic flight. Further complicating the characterization of the aerodynamics and the resultant vehicle controllability is the interaction of the vehicle flowfield with the plumes of the two solid propellant motors that provide attitude control and the main propulsive impulse for the LAV. These interactions are a function of flight parameters such as Mach number, altitude, dynamic pressure, vehicle attitude, as well as parameters relating to the operation of the motors themselves - either as a function of time for the AM, or as a result of the flight control system requests for control torque from the ACM. This paper discusses the computational aerodynamic modeling of the aerodynamic interaction caused by main abort motor and the attitude control motor of the MPCV LAV, showing the effects of these interactions on vehicle controllability.

An approach to simultaneous control of trajectory and interaction forces in dual-arm configurations

NASA Technical Reports Server (NTRS)

Yun, Xiaoping; Kumar, Vijay R.

1991-01-01

An approach to the control of constrained dynamic systems such as multiple arm systems, multifingered grippers, and walking vehicles is described. The basic philosophy is to utilize a minimal set of inputs to control the trajectory and the surplus input to control the constraint or interaction forces and moments in the closed chain. A dynamic control model for the closed chain is derived that is suitable for designing a controller in which the trajectory and the interaction forces and moments are explicitly controlled. Nonlinear feedback techniques derived from differential geometry are then applied to linearize and decouple the nonlinear model. These ideas are illustrated through a planar example in which two arms are used for cooperative manipulation. Results from a simulation are used to illustrate the efficacy of the method.

Analysis, preliminary design and simulation systems for control-structure interaction problems

NASA Technical Reports Server (NTRS)

Park, K. C.; Alvin, Kenneth F.

1991-01-01

Software aspects of control-structure interaction (CSI) analysis are discussed. The following subject areas are covered: (1) implementation of a partitioned algorithm for simulation of large CSI problems; (2) second-order discrete Kalman filtering equations for CSI simulations; and (3) parallel computations and control of adaptive structures.

Persistence Temperament Associated with Children Playing Math Games between Touch Panel and Embodied Interaction

ERIC Educational Resources Information Center

Jong, J-T.; Hong, J-C.; Yen, C-Y.

2013-01-01

The different designs of various interactive gaming controllers affect posture and muscle loading of the body. It is assumed that prolonged exposure to the interactive gaming controllers can affect the effectiveness in using the game for the purposes of learning. This study explores the differences in behavioural responses among students with…

The interaction of amylin with other hormones in the control of eating.

PubMed

Lutz, T A

2013-02-01

Twenty years of research established amylin as an important control of energy homeostasis. Amylin controls nutrient and energy fluxes by reducing energy intake, by modulating nutrient utilization via an inhibition of postprandial glucagon secretion and by increasing energy disposal via a prevention of compensatory decreases of energy expenditure in weight reduced individuals. Like many other gastrointestinal hormones, amylin is secreted in response to meals and it reduces eating by promoting meal-ending satiation. Not surprisingly, amylin interacts with many of these hormones to control eating. These interactions seem to occur at different levels because amylin seems to mediate the eating inhibitory effect of some of these gastrointestinal hormones, and the combination of some of these hormones seems to lead to a stronger reduction in eating than single hormones alone. Amylin's effect on eating is thought to be mediated by a stimulation of specific amylin receptors in the area postrema. Secondary brain sites that were defined to mediate amylin action - and hence potential additional sites of interaction with other hormones - include the nucleus of the solitary tract, the lateral parabrachial nucleus, the lateral hypothalamic area and other hypothalamic nuclei. The focus of this review is to summarize the current knowledge of amylin interactions in the control of eating. In most cases, these interactions have only been studied at a descriptive rather than a mechanistic level and despite the clear knowledge on primary sites of amylin action, the interaction sites between amylin and other hormones are often unknown. © 2012 Blackwell Publishing Ltd.

Gene × environment interaction studies have not properly controlled for potential confounders: the problem and the (simple) solution.

PubMed

Keller, Matthew C

2014-01-01

Candidate gene × environment (G × E) interaction research tests the hypothesis that the effects of some environmental variable (e.g., childhood maltreatment) on some outcome measure (e.g., depression) depend on a particular genetic polymorphism. Because this research is inherently nonexperimental, investigators have been rightly concerned that detected interactions could be driven by confounders (e.g., ethnicity, gender, age, socioeconomic status) rather than by the specified genetic or environmental variables per se. In an attempt to eliminate such alternative explanations for detected G × E interactions, investigators routinely enter the potential confounders as covariates in general linear models. However, this practice does not control for the effects these variables might have on the G × E interaction. Rather, to properly control for confounders, researchers need to enter the covariate × environment and the covariate × gene interaction terms in the same model that tests the G × E term. In this manuscript, I demonstrate this point analytically and show that the practice of improperly controlling for covariates is the norm in the G × E interaction literature to date. Thus, many alternative explanations for G × E findings that investigators had thought were eliminated have not been. © 2013 Society of Biological Psychiatry Published by Society of Biological Psychiatry All rights reserved.

Evaluation of an eye-pointer interaction device for human-computer interaction.

PubMed

Cáceres, Enrique; Carrasco, Miguel; Ríos, Sebastián

2018-03-01

Advances in eye-tracking technology have led to better human-computer interaction, and involve controlling a computer without any kind of physical contact. This research describes the transformation of a commercial eye-tracker for use as an alternative peripheral device in human-computer interactions, implementing a pointer that only needs the eye movements of a user facing a computer screen, thus replacing the need to control the software by hand movements. The experiment was performed with 30 test individuals who used the prototype with a set of educational videogames. The results show that, although most of the test subjects would prefer a mouse to control the pointer, the prototype tested has an empirical precision similar to that of the mouse, either when trying to control its movements or when attempting to click on a point of the screen.

Dark soliton dynamics and interactions in continuous-wave-induced lattices.

PubMed

Tsopelas, Ilias; Kominis, Yannis; Hizanidis, Kyriakos

2007-10-01

The dynamics of dark spatial soliton beams and their interaction under the presence of a continuous wave (CW), which dynamically induces a photonic lattice, are investigated. It is shown that appropriate selection of the characteristic parameters of the CW result in controllable steering of a single soliton as well as controllable interaction between two solitons. Depending on the CW parameters, the soliton angle of propagation can be changed drastically, while two-soliton interaction can be either enhanced or reduced, suggesting a reconfigurable soliton control mechanism. Our analytical approach, based on the variational perturbation method, provides a dynamical system for the dark soliton evolution parameters. Analytical results are shown in good agreement with direct numerical simulations.