Domain Interface Research Articles

A General High-Order Multi-Domain Hybrid DG/WENO-FD Method for Hyperbolic Conservation Laws

In this paper, a general high-order multi-domain hybrid DG/WENO-FD method, which couples a p th -order (p � 3) DG method and a q th -order (q � 3) WENO-FD scheme, is developed. There are two possible coupling approaches at the domain interface, one is non-conservative, the other is conservative. The non-conservative coupling approach can preserve optimal order of accuracy and the local conservative error is proved to be upmost third order. As for the conservative coupling approach, accuracy analysis shows the forced conservation strategy at the coupling interface deteriorates the accuracy locally to firstorder accuracy at the ‘coupling cell’. A numerical experiments of numerical stability is also presented for the non-conservative and conservative coupling approaches. Several numerical results are presented to verify the theoretical analysis results and demonstrate the performance of the hybrid DG/WENO-FD solver.

Local Peltier-effect-induced reversible metal–insulator transition in VO2 nanowires

We report anomalous resistance leaps and drops in VO2 nanowires with operating current density and direction, showing reversible and nonvolatile switching. This event is associated with the metal–insulator phase transition (MIT) of local nanodomains with coexistence states of metallic and insulating phases induced by thermoelectric cooling and heating effects. Because the interface of metal and insulator domains has much different Peltier coefficient, it is possible that a significant Peltier effect would be a source of the local MIT. This operation can be realized by one-dimensional domain configuration in VO2 nanowires because one straight current path through the electronic domain-interface enables theoretical control of thermoelectric effects. This result will open a new method of reversible control of electronic states in correlated electron materials.

A practical automated timing and physical design implementation methodology for the synchronous asynchronous interface and multi-voltage domain in high-speed synthesis

In a high-speed synthesis design environment, designers struggle to ensure that multi-clock and multi-power interfaces are designed, placed, connected and timed correctly. Identifying and applying proper timing constraints such as “no cycle stealing” at synchronous and asynchronous domain interfaces in macro synthesis, unit and chip timing are essential. Standard cell library characterization, for multi-power and timing challenges due to an additional delay for level translator circuitry, demand careful implementation in a high-speed synthesis methodology. We propose a pseudo algorithm and methodology for synthesis and timing that will correctly identify synchronous and asynchronous interfaces. Our proposed methodology shows how these interface paths should be excluded from “cycle stealing” and yet, take full advantage of slack borrowing for the rest of the design. We find ∼28% and ∼80% timing path improvement in two of the units for IBM's Power8TM (P8) microprocessor. As an alternative to high-effort custom design, we develop a synthesis-based physical design methodology that incorporates the use of a level translator, enabling designers to address major issues that encompass dual-voltage solutions in high-speed design. We find ∼50% physical design effort savings using this methodology. P8 is a 12-core, 649 mm2, 4.2B transistor chip fabricated in IBM's 22-nm Silicon On Insulator (SOI) technology, which is fully functional to support a wide range of high performing systems with an operating frequency greater than 4.5GHz.

Influence of the Fe-Co ratio on the exchange coupling in TbFeCo/[Co/Pt] heterostructures

We report on a systematic study of exchange coupled heterostructures, consisting of ferromagnetic [Co/Pt] multilayers and ferrimagnetic (FI) $\mathrm{T}{\mathrm{b}}_{x}\mathrm{F}{\mathrm{e}}_{100\ensuremath{-}x\ensuremath{-}y}\mathrm{C}{\mathrm{o}}_{y}$ (20 nm) alloy thin films with varying composition exhibiting strong perpendicular magnetic anisotropy. In particular, the impact of the Tb content and ratio of Fe and Co of the amorphous FI alloy on the exchange interaction at the interface was investigated. In this paper, the magnetic properties of single ternary TbFeCo thin films were analyzed in a broad composition range and compared to coupled TbFeCo/[Co/Pt] heterostructures. While a rather linear dependence of the exchange coupling strength was observed for Fe/Co-dominated ferrimagnets with increasing amount of Co, a nonlinear behavior is observed for Tb-dominated alloys. The latter behavior is governed by the variation of the exchange stiffness of the ferrimagnet. Additionally, by using element-specific x-ray magnetic circular dichroism measurements, the thickness of the interface domain wall (IDW) in the ferrimagnet, which is formed during the reversal of the ferromagnet, can be extracted. An inverse correlation between the IDW thickness and the exchange coupling strength at the interface was deduced.

Flavonolignans As a Novel Class of Sodium Pump Inhibitors.

We examined the inhibitory effects of three flavonolignans and their dehydro- derivatives, taxifolin and quercetin on the activity of the Na+/K+-ATPase (NKA). The flavonolignans silychristin, dehydrosilychristin and dehydrosilydianin inhibited NKA with IC50 of 110 ± 40 μM, 38 ± 8 μM, and 36 ± 14 μM, respectively. Using the methods of molecular modeling, we identified several possible binding sites for these species on NKA and proposed the possible mechanisms of inhibition. The binding to the extracellular- or cytoplasmic C-terminal sites can block the transport of cations through the plasma membrane, while the binding on the interface of cytoplasmic domains can inhibit the enzyme allosterically. Fluorescence spectroscopy experiments confirmed the interaction of these three species with the large cytoplasmic segment connecting transmembrane helices 4 and 5 (C45). The flavonolignans are distinct from the cardiac glycosides that are currently used in NKA treatment. Because their binding sites are different, the mechanism of inhibition is different as well as the range of active concentrations, one can expect that these new NKA inhibitors would exhibit also a different biomedical actions than cardiac glycosides.

Protons Potentiate GluN1/GluN3A Currents by Attenuating Their Desensitisation.

N-methyl-D-aspartate (NMDA) receptors are glutamate- and glycine-gated channels composed of two GluN1 and two GluN2 or/and GluN3 subunits. GluN3A expression is developmentally regulated, and changes in this normal pattern of expression, which occur in several brain disorders, alter synaptic maturation and function by unknown mechanisms. Uniquely within the NMDA receptor family, GluN1/GluN3 receptors produce glycine-gated deeply desensitising currents that are insensitive to glutamate and NMDA; these currents remain poorly characterised and their cellular functions are unknown. Here, we show that extracellular acidification strongly potentiated glycine-gated currents from recombinant GluN1/GluN3A receptors, with half-maximal effect in the physiologic pH range. This was largely due to slower current desensitisation and faster current recovery from desensitisation, and was mediated by residues facing the heterodimer interface of the ligand-binding domain. Consistent with the observed changes in desensitisation kinetics, acidic shifts increased the GluN1/GluN3A equilibrium current and depolarized the membrane in a glycine concentration-dependent manner. These results reveal novel modulatory mechanisms for GluN1/GluN3A receptors that further differentiate them from the canonical glutamatergic GluN1/GluN2 receptors and provide a new and potent pharmacologic tool to assist the detection, identification, and the further study of GluN1/GluN3A currents in native preparations.

Creation of a Ligand-Dependent Enzyme by Fusing Circularly Permuted Antibody Variable Region Domains

Allosteric control of enzyme activity with exogenous substances has been hard to achieve, especially using antibody domains that potentially allow control by any antigens of choice. Here, in order to attain this goal, we developed a novel antibody variable region format introduced with circular permutations, called Clampbody. The two variable-region domains of the antibone Gla protein (BGP) antibody were each circularly permutated to have novel termini at the loops near their domain interface. Through their attachment to the N- and C-termini of a circularly permutated TEM-1 β-lactamase (cpBLA), we created a molecular switch that responds to the antigen peptide. The fusion protein specifically recognized the antigen, and in the presence of some detergent or denaturant, its catalytic activity was enhanced up to 4.7-fold in an antigen-dependent manner, due to increased resistance to these reagents. Hence, Clampbody will be a powerful tool for the allosteric regulation of enzyme and other protein activities and especially useful to design robust biosensors.

Structure-Based Design of a Br Halogen Bond at the Complex Interface of the Human Placental HtrA1 PDZ Domain with Its Heptapeptide Ligand.

The shock-induced serine protease HtrA1 is a potential regulator of human placenta development during pregnancy. The protein contains a functional PDZ domain that has been solved in complex with a phage display-derived heptapeptide: Asp-6 Ser-5 Arg-4 Ile-3 Trp-2 Trp-1 Val0 . In this study, a rationally designed halogen bond was introduced to the domain-peptide complex based on its NMR structure in solution. We computationally compared the stabilization energies and hindrance effects due to the presence of different halogens X (X = F, Cl, Br, or I), using a hybrid quantum mechanics/molecular mechanics (QM/MM) approach, and found that the Br atom could considerably promote the peptide binding free energy (ΔΔG = -5.2 kcal/mol). Fluorescence assays confirmed that the peptide affinity to the HtrA1 PDZ domain was improved by approximately sevenfold upon bromination. Structural analysis identified a geometrically perfect halogen bond between the Br atom of the peptide Trp-1 residue and the carbonyl O atom of the HtrA1 Ile385 residue, with a bond length and an interaction energy of d = 3.20 Å and ΔE = -3.7 kcal/mol, respectively.

Signal Transduction at the Domain Interface of Prokaryotic Pentameric Ligand-Gated Ion Channels

Pentameric ligand-gated ion channels are activated by the binding of agonists to a site distant from the ion conduction path. These membrane proteins consist of distinct ligand-binding and pore domains that interact via an extended interface. Here, we have investigated the role of residues at this interface for channel activation to define critical interactions that couple conformational changes between the two structural units. By characterizing point mutants of the prokaryotic channels ELIC and GLIC by electrophysiology, X-ray crystallography and isothermal titration calorimetry, we have identified conserved residues that, upon mutation, apparently prevent activation but not ligand binding. The positions of nonactivating mutants cluster at a loop within the extracellular domain connecting β-strands 6 and 7 and at a loop joining the pore-forming helix M2 with M3 where they contribute to a densely packed core of the protein. An ionic interaction in the extracellular domain between the turn connecting β-strands 1 and 2 and a residue at the end of β-strand 10 stabilizes a state of the receptor with high affinity for agonists, whereas contacts of this turn to a conserved proline residue in the M2-M3 loop appear to be less important than previously anticipated. When mapping residues with strong functional phenotype on different channel structures, mutual distances are closer in conducting than in nonconducting conformations, consistent with a potential role of contacts in the stabilization of the open state. Our study has revealed a pattern of interactions that are crucial for the relay of conformational changes from the extracellular domain to the pore region of prokaryotic pentameric ligand-gated ion channels. Due to the strong conservation of the interface, these results are relevant for the entire family.

Substructural interface force identification with limited vibration measurements

This paper presents a dynamic substructural interface force identification approach with limited vibration measurements from the target substructure. The substructure is considered to be subjected to both the external excitation forces at some specific degrees-of-freedom (DOFs) within the substructure and the interface forces at interface DOFs. The interface force time histories are represented as orthogonal polynomials and their discrete Fourier transform is expressed in the matrix multiplication form which transforms the time domain interface forces into their Fourier transforms in the frequency domain. A frequency-time domain method is used to formulate the identification equation based on the relationship between two sets of response vectors in a substructure. The coefficients of orthogonal functions are identified with the Tikhonov regularization technique. Numerical studies on a seven-storey plane frame structure are conducted to investigate the accuracy and efficiency of the proposed substructure interface force identification approach.

TRPC6 G757D Loss-of-Function Mutation Associates with FSGS.

FSGS is a CKD with heavy proteinuria that eventually progresses to ESRD. Hereditary forms of FSGS have been linked to mutations in the transient receptor potential cation channel, subfamily C, member 6 (TRPC6) gene encoding a nonselective cation channel. Most of these TRPC6 mutations cause a gain-of-function phenotype, leading to calcium-triggered podocyte cell death, but the underlying molecular mechanisms are unclear. We studied the molecular effect of disease-related mutations using tridimensional in silico modeling of tetrameric TRPC6. Our results indicated that G757 is localized in a domain forming a TRPC6-TRPC6 interface and predicted that the amino acid exchange G757D causes local steric hindrance and disruption of the channel complex. Notably, functional characterization of model interface domain mutants suggested a loss-of-function phenotype. We then characterized 19 human FSGS-related TRPC6 mutations, the majority of which caused gain-of-function mutations. However, five mutations (N125S, L395A, G757D, L780P, and R895L) caused a loss-of-function phenotype. Coexpression of wild-type TRPC6 and TRPC6 G757D, mimicking heterozygosity observed in patients, revealed a dominant negative effect of TRPC6 G757D. Our comprehensive analysis of human disease-causing TRPC6 mutations reveals loss of TRPC6 function as an additional concept of hereditary FSGS and provides molecular insights into the mechanism responsible for the loss-of-function phenotype of TRPC6 G757D in humans.

Frequency and intensity of palaeofloods at the interface of Atlantic and Mediterranean climate domains

Abstract. Mediterranean climatic influences was explored by studying a lake sequence (Lake Foréant) of the Western European Alps. High-resolution sedimentological and geochemical analysis revealed 171 event layers, 168 of which result from past flood events over the last millennium. The layer thickness was used as a proxy of intensity of past floods. Because the Foréant palaeoflood record is in agreement with the documented variability of historical floods resulting from local and mesoscale, summer-to-autumn convective events, it is assumed to highlight changes in flood frequency and intensity related to such events typical of both Atlantic (local events) and Mediterranean (mesoscale events) climatic influences. Comparing the Foréant record with other Atlantic-influenced and Mediterranean-influenced regional flood records highlights a common feature in all flood patterns that is a higher flood frequency during the cold period of the Little Ice Age (LIA, AD 1300–1900). In contrast, high-intensity flood events are apparent during both the cold LIA and the warm Medieval Climate Anomaly (MCA, AD 950–1250). However, there is a tendency towards higher frequencies of high-intensity flood events during the warm MCA. The MCA extremes could mean that under the global warming scenario, we might see an increase in intensity (not in frequency). However, the flood frequency and intensity in the course of the 20th century warming trend did not change significantly. Uncertainties in future evolution of flood intensity lie in the interpretation of the lack of 20th century extremes (transition or stable?) and the different climate forcing factors between the two periods (greenhouse gases vs. solar and/or volcanic eruptions).

A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding.

Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration.

Positive Allosteric Modulators of GluN2A-Containing NMDARs with Distinct Modes of Action and Impacts on Circuit Function

To enhance physiological function of NMDA receptors (NMDARs), we identified positive allosteric modulators (PAMs) of NMDARs with selectivity for GluN2A subunit-containing receptors. X-ray crystallography revealed a binding site at the GluN1-GluN2A dimer interface of the extracellular ligand-binding domains (LBDs). Despite the similarity between the LBDs of NMDARs and AMPA receptors (AMPARs), GluN2A PAMs with good selectivity against AMPARs were identified. Potentiation was observed with recombinant triheteromeric GluN1/GluN2A/GluN2B NMDARs and with synaptically activated NMDARs in brain slices from wild-type (WT), but not GluN2A knockout (KO), mice. Individual GluN2A PAMs exhibited variable degrees of glutamate (Glu) dependence, impact on NMDAR Glu EC50, and slowing of channel deactivation. These distinct PAMs also exhibited differential impacts during synaptic plasticity induction. The identification of a new NMDAR modulatory site and characterization of GluN2A-selective PAMs provide powerful molecular tools to dissect NMDAR function and demonstrate the feasibility of a therapeutically desirable type of NMDAR enhancement.

Evidence for Dual Binding Sites for 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) in Insect Sodium Channels

1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), the first organochlorine insecticide, and pyrethroid insecticides are sodium channel agonists. Although the use of DDT is banned in most of the world due to its detrimental impact on the ecosystem, indoor residual spraying of DDT is still recommended for malaria control in Africa. Development of resistance to DDT and pyrethroids is a serious global obstacle for managing disease vectors. Mapping DDT binding sites is necessary for understanding mechanisms of resistance and modulation of sodium channels by structurally different ligands. The pioneering model of the housefly sodium channel visualized the first receptor for pyrethroids, PyR1, in the II/III domain interface and suggested that DDT binds within PyR1. Previously, we proposed the second pyrethroid receptor, PyR2, at the I/II domain interface. However, whether DDT binds to both pyrethroid receptor sites remains unknown. Here, using computational docking of DDT into the Kv1.2-based mosquito sodium channel model, we predict that two DDT molecules can bind simultaneously within PyR1 and PyR2. The bulky trichloromethyl group of each DDT molecule fits snugly between four helices in the bent domain interface, whereas two p-chlorophenyl rings extend into two wings of the interface. Model-driven mutagenesis and electrophysiological analysis confirmed these propositions and revealed 10 previously unknown DDT-sensing residues within PyR1 and PyR2. Our study proposes a dual DDT-receptor model and provides a structural background for rational development of new insecticides.

Structure and Function of a Sodium and Proton Coupled Nucleobase/Ascorbate Transporter

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, concentrative vitamin C uptake by members of the NAT family protein is driven by the Na+ gradient, while in bacteria the H+ gradient drives the uptake of nucleobases. Little is known about the mechanism of substrate binding and translocation. Here we report the structure and function of a NAT protein, PaaTCp, from the bacterium Colwellia psychrerythraea. PaaTCp transports purine bases but not pyrimidine bases, and the transport is driven by a H+ gradient. Remarkably, PaaTCp also transports vitamin C in Na+-dependent manner. The structure of PaaTCp was solved by X-ray crystallography to 2.85 A resolution. PaaTCp forms a homodimer, and each protomer is composed of two domains, an interface domain and a transport domain. The interface domain is formed by six helices arranged in a panel-like configuration, while the transport domain is more compact with eight helices. A purine base is present in the structure and defines the location of the substrate binding site. Mutations to a conserved aspartate residue near the substrate binding site suggests that it is likely involved in mediating the H+- and Na+-dependency. Comparing the PaaTCp structure to other structures of NAT family homologs led us to propose a transport model in which conformational changes of the transport domain enable substrate translocation across the membrane.

Mutational Constraints on Local Unfolding Inhibit the Rheological Adaptation of von Willebrand Factor

Unusually large von Willebrand factor (VWF), the first responder to vascular injury in primary hemostasis, is designed to capture platelets under the high shear stress of rheological blood flow. In type 2M von Willebrand disease, two rare mutations (G1324A and G1324S) within the platelet GPIbα binding interface of the VWF A1 domain impair the hemostatic function of VWF. We investigate structural and conformational effects of these mutations on the A1 domain's efficacy to bind collagen and adhere platelets under shear flow. These mutations enhance the thermodynamic stability, reduce the rate of unfolding, and enhance the A1 domain's resistance to limited proteolysis. Collagen binding affinity is not significantly affected indicating that the primary stabilizing effect of these mutations is to diminish the platelet binding efficiency under shear flow. The enhanced stability stems from the steric consequences of adding a side chain (G1324A) and additionally a hydrogen bond (G1324S) to His(1322) across the β2-β3 hairpin in the GPIbα binding interface, which restrains the conformational degrees of freedom and the overall flexibility of the native state. These studies reveal a novel rheological strategy in which the incorporation of a single glycine within the GPIbα binding interface of normal VWF enhances the probability of local unfolding that enables the A1 domain to conformationally adapt to shear flow while maintaining its overall native structure.

Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State

GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca(2+)-sensitive activation of retinal guanylyl cyclase 1 (RetGC1). We present NMR resonance assignments, residual dipolar coupling data, functional analysis, and a structural model of GCAP1 mutant (GCAP1(V77E)) in the Ca(2+)-free/Mg(2+)-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca(2+)-dependent differences for residues 170-174. An NMR-derived model of GCAP1(V77E) contains Mg(2+) bound at EF2 and looks similar to Ca(2+) saturated GCAP1 (root mean square deviations = 2.0 Å). Ca(2+)-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164-174 called the Ca(2+) switch helix). Ca(2+)-induced shortening of the Ca(2+) switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca(2+) switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca(2+) binding affinity and abolished RetGC1 activation. We conclude that Ca(2+)-dependent conformational changes in the Ca(2+) switch helix are important for activating RetGC1 and provide further support for a Ca(2+)-myristoyl tug mechanism.

Crystal structure of the flavoenzyme PA4991 from Pseudomonas aeruginosa.

The locus PA4991 in Pseudomonas aeruginosa encodes an open reading frame that has been identified as essential for the virulence and/or survival of this pathogenic organism in the infected host. Here, it is shown that this gene encodes a monomeric FAD-binding protein of molecular mass 42.2 kDa. The structure of PA4991 was determined by a combination of molecular replacement using a search model generated with Rosetta and phase improvement by a low-occupancy heavy-metal derivative. PA4991 belongs to the GR2 family of FAD-dependent oxidoreductases, comprising an FAD-binding domain typical of the glutathione reductase family and a second domain dominated by an eight-stranded mixed β-sheet. Most of the protein-FAD interactions are via the FAD-binding domain, but the isoalloxazine ring is located at the domain interface and interacts with residues from both domains. A comparison with the structurally related glycine oxidase and glycerol-3-phosphate dehydrogenase shows that in spite of very low amino-acid sequence identity (<18%) several active-site residues involved in substrate binding in these enzymes are conserved in PA4991. However, enzymatic assays show that PA4991 does not display amino-acid oxidase or glycerol-3-phosphate dehydrogenase activities, suggesting that it requires different substrates for activity.

Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1.

C2 domains are widespread motifs that often serve as Ca(2+)-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca(2+) sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca(2+), and shifted the Ca(2+) dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation-secretion coupling.