Asymmetric Trimers Research Articles

Reconfigurable Assembly of Planar Colloidal Molecules via Chemical Reaction and Electric Polarization.

Colloidal molecules, ordered structures assembled from micro- and nanoparticles, serve as a valuable model for understanding the behavior of real molecules and for constructing materials with tunable properties. In this work, we introduce a universal strategy for assembling colloidal molecules consisting of a central active particle surrounded by several passive particles as ligands. During the assembly process, active particles attract the surrounding passive particles through phoresis and osmosis resulting from the chemical reactions on the surface of the active particles, while passive particles repel each other due to the electric polarization induced by an alternating current (AC) electric field. By carefully selecting particles of varying structures and sizes, we have assembled colloidal molecules of symmetric and asymmetric dimers, trimers, and multimers. Furthermore, the coordination number of these colloidal molecules can be regulated in real time and insitu by tuning the interaction forces between the constituent particles. Brownian dynamics simulations reproduced the formation of the colloidal molecules and validated that the self-assembly arises from chemically induced attraction and electrical dipolar repulsion. This strategy for reconfigurable colloidal assemblies poses the potential for designing adaptive micro-nanomachines.

HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD41–4. Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes5–13. Here we used cryo-electron tomography to monitor Env–CD4 interactions at the membrane–membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env–CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env–CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design.

Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases.

Members of the 4-oxalocrotonate tautomerase (4-OT) subgroup in the tautomerase superfamily (TSF) are constructed from a single β-α-β unit and form homo- or heterohexamers, whereas those of the other four subgroups are composed of two consecutively joined β-α-β units and form trimers. A subset of sequences, double the length of the short 4-OTs, is found in the 4-OT subgroup. These "fused" 4-OTs form a separate subgroup that connects to the short 4-OTs in a sequence similarity network (SSN). The fused gene can be a template for the other four subgroups, resulting in the diversification of activity. Analysis of the SSN shows that multiple nodes in the fused 4-OTs connect to five linker nodes, which in turn connect to the short 4-OTs. Some fused 4-OTs are symmetric trimers and others are asymmetric trimers. The origin of this asymmetry was investigated by subjecting the sequences in three linker nodes and a closely associated fourth node to kinetic, mutagenic, and structural analyses. The results show that each sequence corresponds to the α- or β-subunit of a heterohexamer that functions as a 4-OT. Mutagenesis indicates that the key residues in both are αPro1 and βArg-11, like that of a typical 4-OT. Crystallographic analysis shows that both heterohexamers are asymmetric, where one heterodimer is flipped 180° relative to the other two heterodimers. The fusion of two subunits (α and β) of one asymmetric heterohexamer generates an asymmetric trimer with 4-OT activity. Hence, asymmetry can be introduced at the heterohexamer level and then retained in the fused trimers.

Symmetry of 4-Oxalocrotonate Tautomerase Trimers Influences Unfolding and Fragmentation in the Gas Phase.

The recent discovery of asymmetric arrangements of trimers in the tautomerase superfamily (TSF) adds structural diversity to this already mechanistically diverse superfamily. Classification of asymmetric trimers has previously been determined using X-ray crystallography. Here, native mass spectrometry (MS) and ultraviolet photodissociation (UVPD) are employed as an integrated strategy for more rapid and sensitive differentiation of symmetric and asymmetric trimers. Specifically, the unfolding of symmetric and asymmetric trimers initiated by collisional heating was probed using UVPD, which revealed unique gas-phase unfolding pathways. Variations in UVPD patterns from native-like, compact trimeric structures to unfolded, extended conformations indicate a rearrangement of higher-order structure in the asymmetric trimers that are believed to be stabilized by salt-bridge triads, which are absent from the symmetric trimers. Consequently, the symmetric trimers were found to be less stable in the gas phase, resulting in enhanced UVPD fragmentation overall and a notable difference in higher-order re-structuring based on the extent of hydrogen migration of protein fragments. The increased stability of the asymmetric trimers may justify their evolution and concomitant diversification of the TSF. Facilitating the classification of TSF members as symmetric or asymmetric trimers assists in delineating the evolutionary history of the TSF.

Asymmetric Structures and Conformational Plasticity of the Uncleaved Full-Length Human Immunodeficiency Virus Envelope Glycoprotein Trimer.

The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] is produced by cleavage of a conformationally flexible gp160 precursor. gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pretriggered, "closed" (state 1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more "open" Env conformations (states 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and cross-linking decreased the exposure of pNAb epitopes on cell surface gp160; however, after detergent solubilization, cross-linked and BMS-806-treated gp160 sampled non-state-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume state-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a state-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage for a persistent virus like HIV-1.

Chiral Polymorphic Hydrazine-based Asymmetric Liquid Crystal Trimers with Resorcinol as Linking Group.

This is the first report on chiral polymorphic hydrazine-based asymmetric liquid crystal trimers, 1-[4'-(4''-(5-Cholesteryloxy)carbonyl)butyloxy]-3-[N-benzylideneoxy-N'-(4'''-decyloxybenzylidene) hydrazine] butyloxybenzenes, and 1-[4'-(4''-(10-cholesteryloxy)carbonyl)nonyloxy]-3-[N-benzylideneoxy-N'-(4'''- decyloxybenzylidene)hydrazine]butyloxybenzenes., in which the hydrazine and cholesterol arms were connected via two flexible methylene spacers (n = 3-12 units and m = 4 or 9, respectively) to the central resorcinol core. FT-IR, 1D and 2D NMR spectroscopy, and CHN microanalysis were used to elucidate the structures of the trimers. Differential scanning calorimetry, polarizing optical microscopy and X-ray diffraction were used to study the transitional and phase properties of the trimers, which were length and spacer parity dependent. Trimers with short spacer length in the cholesteryl arm, m = 4 showed an interesting phase sequence of BP/N*-TGBA*-SmA*. The TGBA* phase was sensitive to spacer length as it was only observed in trimers with short ester linkage. For the long analogues, m = 9, characteristic visible reflection and a much simpler phase sequence with only N* and SmA* phases were observed. The X-ray diffraction measurements revealed that layer periodicities of the SmA* phase were approximately half the estimated all-trans molecular length (d/L ≈ 0.44-0.52), thus suggesting that the molecules are either strongly intercalated or bent.

Chiral Polymorphic Hydrazine-based Asymmetric Liquid Crystal Trimers with Resorcinol as Linking Group

Chiral Polymorphic Hydrazine-based Asymmetric Liquid Crystal Trimers with Resorcinol as Linking Group

Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.

Structural Basis for the Asymmetry of a 4-Oxalocrotonate Tautomerase Trimer.

Tautomerase superfamily (TSF) members are constructed from a single β-α-β unit or two consecutively joined β-α-β units. This pattern prevails throughout the superfamily consisting of more than 11000 members where homo- or heterohexamers are localized in the 4-oxalocrotonate tautomerase (4-OT) subgroup and trimers are found in the other four subgroups. One exception is a subset of sequences that are double the length of the short 4-OTs in the 4-OT subgroup, where the coded proteins form trimers. Characterization of two members revealed an interesting dichotomy. One is a symmetric trimer, whereas the other is an asymmetric trimer. One monomer is flipped 180° relative to the other two monomers so that three unique protein-protein interfaces are created that are composed of different residues. A bioinformatics analysis of the fused 4-OT subset shows a further division into two clusters with a total of 133 sequences. The analysis showed that members of one cluster (86 sequences) have more salt bridges if the asymmetric trimer forms, whereas the members of the other cluster (47 sequences) have more salt bridges if the symmetric trimer forms. This hypothesis was examined by the kinetic and structural characterization of two proteins within each cluster. As predicted, all four proteins function as 4-OTs, where two assemble into asymmetric trimers (designated R7 and F6) and two form symmetric trimers (designated W0 and Q0). These findings can be extended to the other sequences in the two clusters in the fused 4-OT subset, thereby annotating their oligomer properties and activities.

Integrated structural modeling and super-resolution imaging resolve GPCR oligomers.

Formation of G protein-coupled receptors (GPCRs) dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. For the Class A/rhodopsin subfamily of glycoprotein hormone receptors (GpHRs), di/oligomerization has been demonstrated to play a significant role in regulating its signaling activity at a cellular and physiological level and even pathophysiologically. Here we will describe and discuss the developments in our understanding of GPCR oligomerization, in both health and disease, from the study of this unique and complex subfamily of GPCRs with light on the luteinizing hormone receptor (LHR). Focus will be put on the results of an approach relying on the combination of atomistic modeling by protein-protein docking with super-resolution imaging. The latter could resolve single LHR molecules to ~8nm resolution in functional asymmetric dimers and oligomers, using dual-color photoactivatable dyes and localization microscopy (PD-PALM). Structural modeling of functionally asymmetric LHR trimers and tetramers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.

Chiral Recognition of Hexahelicene on a Surface via the Forming of Asymmetric Heterochiral Trimers.

Chiral recognition among helical molecules is of essential importance in many chemical and biochemical processes. The complexity necessitates investigating manageable model systems for unveiling the fundamental principles of chiral recognition at the molecular level. Here, we reported chiral recognition in the self-assembly of enantiopure and racemic hexahelicene on a Au(111) surface. Combing scanning tunneling microscopy (STM) and atomic force microscopy (AFM) measurements, the asymmetric heterochiral trimers were observed as a new type of building block in racemic helicene self-assembly on Au(111). The intermolecular recognition of the heterochiral trimer was investigated upon manual separation so that the absolute configuration of each helicene molecule was unambiguously determined one by one, thus confirming that the trimer was “2+1” in handedness. These heterochiral trimers showed strong stability upon different coverages, which was also supported by theoretical calculations. Our results provide valuable insights for understanding the intermolecular recognition of helical molecules.

Plasmonic Nanolenses: Electrostatic Self-Assembly of Hierarchical Nanoparticle Trimers and Their Response to Optical and Electron Beam Stimuli.

Asymmetric nanoparticle trimers composed of particles with increasing diameter act as "plasmonic lenses" and have been predicted to exhibit ultrahigh confinement of electromagnetic energy in the space between the two smallest particles. Here we present an electrostatic self-assembly approach for creating gold nanoparticle trimers with an assembly yield of over 60%. We demonstrate that the trimer assembly leads to characteristic red-shifts and show the localization of the relevant plasmon modes by means of cathodoluminescence and electron energy loss spectroscopy. The results are analyzed in terms of surface plasmon hybridization.

Receptor Activation of HIV-1 Env Leads to Asymmetric Exposure of the gp41 Trimer.

Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in a nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI.

Understanding Quantum Plasmonics from Time-Dependent Orbital-Free Density Functional Theory

Using time-dependent orbital-free density functional theory, we perform quantum mechanical simulations to understand plasmonic responses in sodium nanoparticle dimers and trimers. The electronic structure, optical absorption, electric field enhancement, and photoinduced tunneling current are examined. For dimers, the maximum field enhancement is reached at a gap distance of 6 A, below which a conductive channel is formed. The tunneling current peaks at the resonant energy of the charge-transfer plasmon (CTP). One such CTP is formed in symmetric linear trimers, and it splits into two in asymmetric linear trimers—one corresponds to a “global” and the other a “local” oscillation as a result of constructive and destructive interferences between the CTPs of the corresponding dimers. The interference leads to “nanofocusing” where the intensity of the “global” mode reaches its maximum while the “local” mode is quenched completely. Similarly, the electric field can be tuned to be localized at one of the two gaps ...

A novel packing arrangement of AcrB in the lipid bilayer membrane

The central component AcrB of the Escherichia coli drug efflux complex AcrA–AcrB–TolC has been extensively investigated by X-ray crystallography of detergent–protein 3-D crystals. In these crystals, AcrB packs as trimers – the functional unit. We visualized the AcrB–AcrB interaction in its native environment by examining E. coli lipid reconstituted 2-D crystals, which were overwhelmingly formed by asymmetric trimers stabilized by strongly-interacting monomers from adjacent trimers. Most interestingly, we observed lattices formed by an arrangement of AcrB monomers distinct from that in traditional trimers. This hitherto unobserved packing, might play a role in the biogenesis of trimeric AcrB.

EIF2B is a decameric guanine nucleotide exchange factor with a γ2ε2 tetrameric core.

eIF2B facilitates and controls protein synthesis in eukaryotes by mediating guanine nucleotide exchange on its partner eIF2. We combined mass spectrometry (MS) with chemical cross-linking, surface accessibility measurements and homology modelling to define subunit stoichiometry and interactions within eIF2B and eIF2. Although it is generally accepted that eIF2B is a pentamer of five non-identical subunits (α–ε), here we show that eIF2B is a decamer. MS and cross-linking of eIF2B complexes allows us to propose a model for the subunit arrangements within eIF2B where the subunit assembly occurs through catalytic γ- and ε-subunits, with regulatory subunits arranged in asymmetric trimers associated with the core. Cross-links between eIF2 and eIF2B allow modelling of interactions that contribute to nucleotide exchange and its control by eIF2 phosphorylation. Finally, we identify that GTP binds to eIF2Bγ, prompting us to propose a multi-step mechanism for nucleotide exchange.

Alternating-access mechanism in conformationally asymmetric trimers of the betaine transporter BetP

Betaine and Na(+) symport has been extensively studied in the osmotically regulated transporter BetP from Corynebacterium glutamicum, a member of the betaine/choline/carnitine transporter family, which shares the conserved LeuT-like fold of two inverted structural repeats. BetP adjusts its transport activity by sensing the cytoplasmic K(+) concentration as a measure for hyperosmotic stress via the osmosensing carboxy-terminal domain. BetP needs to be in a trimeric state for communication between individual protomers through several intratrimeric interaction sites. Recently, crystal structures of inward-facing BetP trimers have contributed to our understanding of activity regulation on a molecular level. Here we report new crystal structures, which reveal two conformationally asymmetric BetP trimers, capturing among them three distinct transport states. We observe a total of four new conformations at once: an outward-open apo and an outward-occluded apo state, and two closed transition states--one in complex with betaine and one substrate-free. On the basis of these new structures, we identified local and global conformational changes in BetP that underlie the molecular transport mechanism, which partially resemble structural changes observed in other sodium-coupled LeuT-like fold transporters, but show differences we attribute to the osmolytic nature of betaine, the exclusive substrate specificity and the regulatory properties of BetP.

Single Molecule Multiphotochromism with Diarylethenes

In single photochromes, the two isomers that are interconverted in photoinduced reactions can serve as on and off states in a molecular switching device. The addition of several photochromic moieties onto a single molecule can allow the processing of more complex logical patterns. For example, an asymmetric triad could, in principle, store a byte, rather than a bit, of data. Because of the potential impact of multiphotochromic molecules in many research areas, over the past decade several groups have synthesized these coupled structures. The targets are easily addressable molecules that display increased contrast between the on and off states and in which all isomers have significantly distinguishable optical signatures. In this Account, we provide an overview of the multiswitchable molecular systems that incorporate at least one diarylethene group, which is the most successful thermally stable (P-type) organic photochrome. Up to this point, most systems have presented significant limitations. First of all, the reversibility of the processes is hindered by several side reactions more frequently than for single photochromes. Second, switching one part of the compound impedes the photoreactivity of other fragments in approximately 50% of the cases, and maximizing the electronic communication increases the probability of partial activity. In addition, most of the few synthesized operative systems only demonstrate cumulative absorption spectra rather than new features. Finally, it is impossible to selectively induce a chosen conversion because one wavelength might trigger several processes. We also emphasize the promising successes of asymmetric diarylethene dimers and trimers and molecules that combine two families of photochromes, such as diarylethene added to fulgimide or phenoxy-naphthacenequinone. In that framework, theoretical simulations offer complementary tools to investigate these structures, both to obtain structure/property relationships and to propose paths for the design of more efficient molecules. However, due to the size of the systems, researchers can only apply semiquantitative models. The investigation of the absorption spectra of the photochromes with time-dependent density functional theory (TD-DFT), the analysis of the topology of the LUMO + n (typically n = 1) of the closed-open hybrid, and an estimate of the steric stress in the hypothetical (ground-state) closed-closed structure serve as a useful combination of parameters to obtain initial insights regarding the photocyclization of the different open diarylethene groups. Nevertheless, because a first-order qualitative approach does not explore the potential energy surface of the photoexcited states, it remains inadequate for the investigation of some molecules.

DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa

DCP1 stimulates the decapping enzyme DCP2, which removes the mRNA 5' cap structure committing mRNAs to degradation. In multicellular eukaryotes, DCP1-DCP2 interaction is stabilized by additional proteins, including EDC4. However, most information on DCP2 activation stems from studies in S. cerevisiae, which lacks EDC4. Furthermore, DCP1 orthologs from multicellular eukaryotes have a C-terminal extension, absent in fungi. Here, we show that in metazoa, a conserved DCP1 C-terminal domain drives DCP1 trimerization. Crystal structures of the DCP1-trimerization domain reveal an antiparallel assembly comprised of three kinked alpha-helices. Trimerization is required for DCP1 to be incorporated into active decapping complexes and for efficient mRNA decapping in vivo. Our results reveal an unexpected connectivity and complexity of the mRNA decapping network in multicellular eukaryotes, which likely enhances opportunities for regulating mRNA degradation.

Managing light polarization via plasmon–molecule interactions within an asymmetric metal nanoparticle trimer

The interaction of light with metal nanoparticles leads to novel phenomena mediated by surface plasmon excitations. In this article we use single molecules to characterize the interaction of surface plasmons with light, and show that such interaction can strongly modulate the polarization of the emitted light. The simplest nanostructures that enable such polarization modulation are asymmetric silver nanocrystal trimers, where individual Raman scattering molecules are located in the gap between two of the nanoparticles. The third particle breaks the dipolar symmetry of the two-particle junction, generating a wavelength-dependent polarization pattern. Indeed, the scattered light becomes elliptically polarized and its intensity pattern is rotated in the presence of the third particle. We use a combination of spectroscopic observations on single molecules, scanning electron microscope imaging, and generalized Mie theory calculations to provide a full picture of the effect of particles on the polarization of the emitted light. Furthermore, our theoretical analysis allows us to show that the observed phenomenon is very sensitive to the size of the trimer particles and their relative position, suggesting future means for precise control of light polarization on the nanoscale.