Asymmetric Dimer Research Articles

Crystal–nematic phase separation in an asymmetric liquid crystal dimer possessing a terminal hydroxyl group

ABSTRACTWe prepared an asymmetric liquid crystal dimer possessing a terminal hydroxyl group, 2-{4-{7-[4-(4-cyanophenyl)phenyloxy]heptyloxy}phenyl}-5-(6-hydroxyhexyloxy)pyrimidine, and investigated the phase transition behaviour using polarising optical microscopy and differential scanning calorimetry. The compound exhibited an enantiotropic nematic phase. On cooling, two distinct domains were formed during the nematic-to-crystal phase transition. During heating, one domain melted at 126.4°C to the N phase, whereas the other domain changed to the N phase at 144.6°C. Therefore, the crystal and nematic phases coexisted at a temperature of 18.2 K. The coexistence behaviour characteristics depend on the cooling rate. We discuss the manner in which intermolecular interactions affect the phase transition behaviour.

Impact of the Nanoscale Gap Morphology on the Plasmon Coupling in Asymmetric Nanoparticle Dimer Antennas.

Coupling of plasmon resonances in metallic gap antennas is of interest for a wide range of applications due to the highly localized strong electric fields supported by these structures, and their high sensitivity to alterations of their structure, geometry, and environment. Morphological alterations of asymmetric nanoparticle dimer antennas with (sub)-nanometer size gaps are assigned to changes of their optical response in correlative dark-field spectroscopy and high-resolution transmission electron microscopy (HR-TEM) investigations. This multimodal approach to investigate individual dimer structures clearly demonstrates that the coupling of the plasmon modes, in addition to well-known parameters such as the particle geometry and the gap size, is also affected by the relative alignment of both nanoparticles. The investigations corroborate that the alignment of the gap forming facets, and with that the gap area, is crucial for their scattering properties. The impact of a flat versus a rounded gap structure on the optical properties of equivalent dimers becomes stronger with decreasing gap size. These results hint at a higher confinement of the electric field in the gap and possibly a different onset of quantum transport effects for flat and rounded gap antennas in corresponding structures for very narrow gaps.

Odd–even effects of an asymmetric dimer on the double-twist structure in an amorphous blue phase

Some chiral mixtures doped with a small amount of an even-membered asymmetric dimer were found to exhibit a phase sequence of I–N*H–BPIII–N*L.

Evolution of magnetization due to asymmetric dimerization: theoretical considerations and application to aberrant oligomers formed by apoSOD1(2SH).

A set of coupled differential equations is presented describing the evolution of magnetization due to an exchange reaction whereby a pair of identical monomers form an asymmetric dimer. In their most general form the equations describe a three-site exchange process that reduces to two-site exchange under certain limiting conditions that are discussed. An application to the study of sparsely populated, transiently formed sets of aberrant dimers, symmetric and asymmetric, of superoxide dismutase is presented. Fits of concentration dependent CPMG relaxation dispersion profiles provide measures of the dimer dissociation constants and both on- and off-rates. Dissociation constants on the order of 70 mM are extracted from fits of the data, with dimeric populations of ∼2% and lifetimes of ∼6 and ∼2 ms for the symmetric and asymmetric complexes, respectively. This work emphasizes the important role that NMR relaxation experiments can play in characterizing very weak molecular complexes that remain invisible to most biophysical approaches.

Unidirectional light scattering with high efficiency at optical frequencies based on low-loss dielectric nanoantennas.

Dielectric nanoparticles offer low optical losses and access to both electric and magnetic Mie resonances. This enables unidirectional scattering along the incident axis of light, owing to the interference between these two resonances. Here we theoretically and experimentally demonstrate that an asymmetric dimer of dielectric nanoparticles can provide unidirectional forward scattering with high efficiency. Theoretical analyses reveal that the dimer configuration can satisfy the first Kerker condition at the resonant peaks of electric and magnetic dipolar modes, therefore showing highly efficient directional forward scattering. The unidirectional forward scattering with high efficiency is confirmed in our experiments using a silicon nanodisk dimer on a transparent substrate. This study will boost the realization of practical applications using low-loss and efficient subwavelength all-dielectric nanoantennas.

Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol

Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD+ as coenzyme. A new X-ray structure was determined at 3.0 Å where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 Å to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 Å from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67.

Switchable directional scattering of electromagnetic radiation with subwavelength asymmetric silicon dimers.

High refractive index dielectric nanoparticles show high promise as a complementary nanophotonics platform due to compared with plasmonic nanostructures low absorption losses and the co-existence of magnetic and electric resonances. Here we explore their use as resonantly enhanced directional scatterers. We theoretically demonstrate that an asymmetric dimer of silicon nanoparticles shows tuneable directional scattering depending on the frequency of excitation. This is due to the interference between electric and magnetic dipoles excited in each nanoparticle, enabling directional control of the scattered light. Interestingly, this control can be achieved regardless of the polarization direction with respect to the dimer axis; however, difference in the polarization can shift the wavelengths at which the directional scattering is achieved. We also explore the application of such an asymmetric nanoantenna as a tuneable routing element in a nanometer scale, suggesting applications in optical nanocircuitry.

Anabaenasp. DyP-type peroxidase is a tetramer consisting of two asymmetric dimers

DyP-type peroxidases are a newly discovered family of heme peroxidases distributed from prokaryotes to eukaryotes. Recently, using a structure-based sequence alignment, we proposed the new classes, P, I and V, as substitutes for classes A, B, C, and D [Arch Biochem Biophys 2015;574:49-55]. Although many class V enzymes from eukaryotes have been characterized, only two from prokaryotes have been reported. Here, we show the crystal structure of one of these two enzymes, Anabaena sp. DyP-type peroxidase (AnaPX). AnaPX is tetramer formed from Cys224-Cys224 disulfide-linked dimers. The tetramer of wild-type AnaPX was stable at all salt concentrations tested. In contrast, the C224A mutant showed salt concentration-dependent oligomeric states: in 600 mM NaCl, it maintained a tetrameric structure, whereas in the absence of salt, it dissociated into monomers, leading to a reduction in thermostability. Although the tetramer exhibits non-crystallographic, 2-fold symmetry in the asymmetric unit, two subunits forming the Cys224-Cys224 disulfide-linked dimer are related by 165° rotation. This asymmetry creates an opening to cavities facing the inside of the tetramer, providing a pathway for hydrogen peroxide access. Finally, a phylogenetic analysis using structure-based sequence alignments showed that class V enzymes from prokaryotes, including AnaPX, are phylogenetically closely related to class V enzymes from eukaryotes.

Structure of Full-Length Human PDGFRβ Bound to Its Activating Ligand PDGF-B as Determined by Negative-Stain Electron Microscopy

Members of the receptor tyrosine kinases (RTKs) regulate important cellular functions such as cell growth and migration, which are key steps in angiogenesis, in organ morphogenesis and in the unregulated states, cancer formation. One long-standing puzzle regarding RTKs centers on how the extracellular domain (ECD), which detects and binds to growth factors, is coupled with the intracellular domain kinase activation. While extensive structural works on the soluble portions of RTKs have provided critical insights into RTK structures and functions, lack of a full-length receptor structure has hindered a comprehensive overview of RTK activation. In this study, we successfully purified and determined a 27-Å-resolution structure of PDGFRβ [a full-length human platelet-derived growth factor receptor], in complex with its ligand PDGF-B. In the ligand-stimulated complex, two PDGFRβs assemble into a dimer via an extensive interface essentially running along the full-length of the receptor, suggesting that the membrane-proximal region, the transmembrane helix and the kinase domain of PDGFRβ are involved in dimerization. Major structural differences are seen between the full-length and soluble ECD structures, rationalizing previous experimental data on how membrane-proximal domains modulate receptor ligand-binding affinity and dimerization efficiency. Also, in contrast to the 2-fold symmetry of the ECD, the intracellular kinase domains adopt an asymmetric dimer arrangement, in agreement with prior observations for the closely related KIT receptor. In essence, the structure provides a first glimpse into how platelet-derived growth factor receptor ECD, upon ligand stimulation, is coupled to its intracellular domain kinase activation.

Double gyroid structures made of asymmetric dimers

ABSTRACTGyroid cubic phases are interesting for both scientists and engineers due to possible applications in electronic devices. New series of dimeric molecules, despite their flexible molecular structure, can display double gyroid cubic phase with Ia3d symmetry and lattice parameter corresponding to double molecular length. The cubic phase is structurally related to columnar phase and both phases often coexist in the same temperature window. Apparently, for studied compounds stronger molecular asymmetry promotes cubic structure. Interestingly, for the examined compounds the transition between two isotropic liquids was observed.

PA62 非対称二量体液晶における結晶-ネマチック相の共存(ソフトマター,ポスター発表,2015年日本液晶学会討論会)

PA62 非対称二量体液晶における結晶-ネマチック相の共存(ソフトマター,ポスター発表,2015年日本液晶学会討論会)

Analysis of the Role of the C-Terminal Tail in the Regulation of the Epidermal Growth Factor Receptor.

The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer.

Structure-Directing Effect of Alkali Metal Cations in New Molybdenum Selenites, Na2Mo2O5(SeO3)2, K2Mo2O5(SeO3)2, and Rb2Mo3O7(SeO3)3.

Both single crystals and pure polycrystalline samples of three new quaternary alkali metal molybdenum selenites, Na2Mo2O5(SeO3)2, K2Mo2O5(SeO3)2, and Rb2Mo3O7(SeO3)3, have been synthesized through hydrothermal and solid-state reactions using A2CO3 (A = Na, K, and Rb), MoO3, and SeO2 as reagents. The frameworks of all three materials consist of both families of second-order Jahn-Teller distortive cations, i.e., the d(0) cation (Mo(6+)) and the lone pair cation (Se(4+)). Although the extent of framework distortions and the resulting occupation sites of alkali metal cations are dissimilar, Na2Mo2O5(SeO3)2 and K2Mo2O5(SeO3)2 exhibit similar three-dimensional networks that are composed of highly asymmetric Mo2O11 dimers and SeO3 polyhedra. Rb2Mo3O7(SeO3)3 reveals a two-dimensional structure that is built with Mo3O15 trimers and SeO3 intralayer linkers. Close structural examinations suggest that the structure-directing effect of alkali metal cations is significant in determining the framework distortions and the dimensions of the molybdenum selenites. UV-vis diffuse reflectance and infrared spectroscopy, thermogravimetric analyses, and ion-exchange reactions are reported, as are out-of-center distortion and dipole moment calculations.

Structure of an intermediate conformer of the spindle checkpoint protein Mad2

The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1-Mad2 core complex. In mitosis, kinetochore-bound Mad1-C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. Here, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2-binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2-C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.

Non‐equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa

In Gram-negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC-OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically in a TriABC-dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC-OpmH. We found that analogous mutations in the α-helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, whereas TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role.

Abstract SY14-02: Regulation of kinase signaling complexes with small molecule inhibitors

Abstract Kinases catalyse the phosphorylation of target substrates on hydroxyl-containing residues as a means to nucleate multi-component complexes or to stabilize unique conformational states. Pseudokinases constitute a subclass of these enzymes that were originally predicted as inactive based on mutations of key conserved active site residues. Like the active kinases, pseudokinases appear to play important functional roles in signal transduction pathways and several pseudokinases have been shown to be important mediators of diseases, including cancer and diabetes. Many pseudokinases share homology inside and outside of the kinase domain with catalytically active kinases. Examples include KSR1 and KSR2, the pseudokinase relatives within the RAF-family of kinases. And Her3, the pseudokinase member of the EGFR family. In both examples, the pseudokinase has been demonstrated to allosterically stimulate the activity of their active ancestral partners through the formation of higher-order complexes. The ability to allosterically modulate active signaling kinases appears to be a dedicated feature of several pseudokinases that overlaps or appears redundant with the ability of active kinases to modulate themselves. For example, KSR forms a high affinity dimer with RAF related kinases and this drives RAF catalytic activation. However, RAF may dimerize with itself to drive its own activation. Clinical inhibitors that bind to RAF are thought to exploit the ability of RAF to regulate itself, leading to activation, rather than inhibition, of RAF when dimeric. Similarly, Her3 dimerizes with EGFR through the formation of an asymmetric dimer, but EGFR appears able to form this dimer in the absence of Her3 as well. In this case, the clinical kinase inhibitor bosutinib, has been shown to function as an agonist through direct modulation of Her3-EGFR complexes. Because pseudokinase function can be redundant with the scaffolding abilities of active kinases, it has been difficult to dissect their precise roles in biology using conventional genetic or biochemical techniques. Small molecules that could antagonize pseudokinase dependent activities would be valuable tools that could be used to functionally annotate the biology and therefore pharmacology of this class of proteins. In particular, direct small molecule antagonists would allow one to disable pseudokinase structure and function on very short timescales and could also provide new leads for therapeutic development. I will describe my laboratories work on developing small molecule modulators of KSR. In particular, I will describe biochemical and cell based approaches used to identify KSR antagonists and furthermore X-ray crystallographic analysis of a lead compound bound to a KSR associated complex. Citation Format: Arvin Dar. Regulation of kinase signaling complexes with small molecule inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY14-02. doi:10.1158/1538-7445.AM2015-SY14-02

Mechanistic Insights into R776H Mediated Activation of Epidermal Growth Factor Receptor Kinase.

The epidermal growth factor receptor (EGFR) kinase is activated by a variety of mutations in human cancers. R776H is one such recurrent mutation (R752H in another numbering system) in the αC-β4 loop of the tyrosine kinase domain that activates EGFR in the absence of the activating EGF ligand. However, the mechanistic details of how R776H contributes to kinase activation are not well understood. Here using cell-based cotransfection assays, we show that the R776H mutation activates EGFR in a dimerization-dependent manner by preferentially adopting the acceptor position in the asymmetric dimer. The acceptor function, but not the donor function, is enhanced for the R776H mutant, supporting the "superacceptor" hypothesis proposed for oncogenic mutations in EGFR. We also find that phosphorylation of monomeric EGFR is increased by R776H mutation, providing insights into EGFR lateral phosphorylation and oligomerization. On the basis of molecular modeling and molecular dynamics simulation, we propose a model in which loss of key autoinhibitory αC-helix capping interaction and alteration of coconserved cis regulatory interactions between the kinase domain and the flanking regulatory segments contribute to mutational activation. Since the R776 equivalent position is mutated in ErbB2 and ErbB4, our studies have implications for understanding kinase mutational activation in other ErbB family members as well.

Electric-field–induced assembly and propulsion of chiral colloidal clusters

Chiral molecules with opposite handedness exhibit distinct physical, chemical, or biological properties. They pose challenges as well as opportunities in understanding the phase behavior of soft matter, designing enantioselective catalysts, and manufacturing single-handed pharmaceuticals. Microscopic particles, arranged in a chiral configuration, could also exhibit unusual optical, electric, or magnetic responses. Here we report a simple method to assemble achiral building blocks, i.e., the asymmetric colloidal dimers, into a family of chiral clusters. Under alternating current electric fields, two to four lying dimers associate closely with a central standing dimer and form both right- and left-handed clusters on a conducting substrate. The cluster configuration is primarily determined by the induced dipolar interactions between constituent dimers. Our theoretical model reveals that in-plane dipolar repulsion between petals in the cluster favors the achiral configuration, whereas out-of-plane attraction between the central dimer and surrounding petals favors a chiral arrangement. It is the competition between these two interactions that dictates the final configuration. The theoretical chirality phase diagram is found to be in excellent agreement with experimental observations. We further demonstrate that the broken symmetry in chiral clusters induces an unbalanced electrohydrodynamic flow surrounding them. As a result, they rotate in opposite directions according to their handedness. Both the assembly and propulsion mechanisms revealed here can be potentially applied to other types of asymmetric particles. Such kinds of chiral colloids will be useful for fabricating metamaterials, making model systems for both chiral molecules and active matter, or building propellers for microscale transport.

Structure of Plasmodium falciparum orotate phosphoribosyltransferase with autologous inhibitory protein-protein interactions.

The most severe form of malaria is caused by the obligate parasite Plasmodium falciparum. Orotate phosphoribosyltransferase (OPRTase) is the fifth enzyme in the de novo pyrimidine-synthesis pathway in the parasite, which lacks salvage pathways. Among all of the malaria de novo pyrimidine-biosynthesis enzymes, the structure of P. falciparum OPRTase (PfOPRTase) was the only one unavailable until now. PfOPRTase that could be crystallized was obtained after some low-complexity sequences were removed. Four catalytic dimers were seen in the asymmetic unit (a total of eight polypeptides). In addition to revealing unique amino acids in the PfOPRTase active sites, asymmetric dimers in the larger structure pointed to novel parasite-specific protein-protein interactions that occlude the catalytic active sites. The latter could potentially modulate PfOPRTase activity in parasites and possibly provide new insights for blocking PfOPRTase functions.

Differential isotopic enrichment to facilitate characterization of asymmetric multimeric proteins using hydrogen/deuterium exchange mass spectrometry.

Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein-ligand and protein-protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched ((15)N-labeled) p51 and unlabeled p66. To enable detection of (15)N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100% (15)N model. Our results demonstrated that (15)N enrichment of p51 did not affect its conformational dynamics compared to unlabeled p51, but (15)N-labeled p51 did show different conformational dynamics than p66 in the RT heterodimer. Differential HDX-MS of isotopically labeled RT in the presence of the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) showed subunit-specific perturbation in the rate of HDX consistent with previously published results and the RT-EFV cocrystal structure.