Asymmetric Dimer Research Articles

Structural basis of Acinetobacter type IV pili targeting by an RNA virus

Acinetobacters pose a significant threat to human health, especially those with weakened immune systems. Type IV pili of acinetobacters play crucial roles in virulence and antibiotic resistance. Single-stranded RNA bacteriophages target the bacterial retractile pili, including type IV. Our study delves into the interaction between Acinetobacter phage AP205 and type IV pili. Using cryo-electron microscopy, we solve structures of the AP205 virion with an asymmetric dimer of maturation proteins, the native Acinetobacter type IV pili bearing a distinct post-translational pilin cleavage, and the pili-bound AP205 showing its maturation proteins adapted to pilin modifications, allowing each phage to bind to one or two pili. Leveraging these results, we develop a 20-kilodalton AP205-derived protein scaffold targeting type IV pili in situ, with potential for research and diagnostics.

The cryo-EM structure of ASK1 reveals an asymmetric architecture allosterically modulated by TRX1.

Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

The cryo-EM structure of ASK1 reveals an asymmetric architecture allosterically modulated by TRX1

Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

RNA-induced Allosteric Coupling Drives Viral Capsid Assembly

Understanding the mechanisms by which single-stranded RNA viruses regulate capsid assembly around their RNA genomes has become increasingly important for the development of both antiviral treatments and drug delivery systems. In this study, we investigate the effects of RNA-induced allostery in a single-stranded RNA virus—Levivirus bacteriophage MS2 assembly—using the computational methods of the Dynamic Flexibility Index and the Dynamic Coupling Index. We demonstrate that not only does asymmetric binding of RNA to a symmetric MS2 coat protein dimer increase the flexibility of the distant FG-loop, inducing a conformational change to an asymmetric dimer, but also RNA binding reorganizes long-distance communications, making all the other positions extremely sensitive to the fluctuation of the ordered FG-loop. Additionally, we find that a point mutation in the FG-loop, W82R, leads to the loss of this asymmetry in communications, likely being a leading cause for assembly-deficient dimers. Lastly, this dominant communication that enhances its dynamic coupling with all the distal positions is not only a property of the dimer but is also exhibited by all the observed capsid intermediates. This strong dynamic coupling allows for unidirectional signal transduction that drives the formation of the experimentally observed capsid intermediates and fully assembled capsid. Published by the American Physical Society 2024

Bimodal surface lattice resonance sensing based on asymmetric metasurfaces

The surface lattice resonance (SLR) is a prominent mechanism to produce ultranarrow spectrum line shape, which can enhance the localized electric field and restrain radiation losses. However, the present research mainly focuses on the single-mode SLR and does not involve the multiplexing and higher-order SLRs. To promote the practicability of SLR, we propose bimodal reflection-type SLRs excited by the natural light based on three kinds of asymmetric optical metasurfaces systemically, which are applied to refractive index sensing with high figures of merit (FoMs) experimentally. The rectangular lattice metasurface breaks the C4 symmetry and produces concurrently (±1, 0) and (0, ±1) order SLRs, with FoMs of 33.50 and 28.85, respectively. In addition, the metasurface composed of two different patches belongs to a spatial multiplexing design and can also realize nearly identical SLR responses. Furthermore, the asymmetric dimer metasurface excites two SLRs with distinct orders meanwhile, where the high-order SLR originates from the trapping of the corresponding Rayleigh anomaly waves. The above-mentioned metasurface designs have flexibility and regularity, whose resonance wavelengths, sensitivities, and bimodal combinations can be attained at will by tuning period lengths, arranging different patches, or forming a dimer meta-atom. The research takes a significant step for bimodal SLR development and application, especially in the sensing field.

Red Circularly Polarized Luminescence from Dimeric H-Aggregates of Acridine Orange by Chiral Induction.

Understanding the mechanism of chirality transfer from a chiral surface to an achiral molecule is essential for designing molecular systems with tunable chiroptical properties. These aspects are explored herein using l- and d-isomers of alkyl valine amphiphiles, which self-assemble in water as nanofibers possessing a negative surface charge. An achiral chromophore, acridine orange, upon electrostatic binding on these surfaces displays mirror-imaged bisignated circular dichroism and red-emitting circularly polarized luminescence signals with a high dissymmetry factor. Experimental and computational investigations establish that the chiroptical properties emerge from surface-bound asymmetric H-type dimers of acridine orange, further supported by fluorescence lifetime imaging studies. Specifically, atomistic molecular dynamics simulations show that the experimentally observed chiral signatures have their origin in van der Waals interactions between acridine orange dimers and the amphiphile head groups as well as in the extent of solvent exposure of the chromophore.

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.

Asymmetric Silicon Dimers Made by Single‐Shot Laser‐Induced Transfer Demultiplex Light of Different Wavelengths

AbstractNanofabrication technologies significantly influence the development of modern optical science. One of such technologies is laser‐induced transfer, which allows the creation of single Mie‐resonant spherical particles on a wide range of substrates. This study shows that this method can provide asymmetric dimers at the output: a single femtosecond pulse being focused on a silicon‐on‐insulator wafer results in appearing two nearly spherical particles of different sizes. The resulting dimers are characterized by scanning electron microscopy, elastic light scattering and Raman spectroscopy to gain insight into their structural properties. Back focal plane imaging and variable‐color evanescent‐wave illumination are then employed to measure the light scattering patterns from isolated dimers. Due to the interference of the excited resonances, the observed patterns are strongly asymmetric in a range of visible wavelengths, which is consistent with theoretical predictions. The results demonstrate the potential of asymmetric silicon dimers made by single‐shot laser‐induced transfer for color routing at visible light.

Different EGF-induced receptor dimer conformations for signaling and internalization.

The structural basis of the activation and internalization of EGF receptors (EGFR) is still a matter of debate despite the importance of this target in cancer treatment. Whether agonists induce dimer formation or act on preformed dimers remains discussed. Here, we provide direct evidence that EGF-induced EGFR dimer formation as best illustrated by the very large increase in FRET between snap-tagged EGFR subunits induced by agonists. We confirm that Erlotinib-related TK (tyrosine kinase) inhibitors also induce dimer formation despite the inactive state of the binding domain. Surprisingly, TK inhibitors do not inhibit EGF-induced EGFR internalization despite their ability to fully block EGFR signaling. Only Erlotinib-related TK inhibitors promoting asymmetric dimers could slow down this process while the lapatinib-related ones have almost no effect. These results reveal that the conformation of the intracellular TK dimer, rather than the known EGFR signaling, is critical for EGFR internalization. These results also illustrate clear differences in the mode of action of TK inhibitors on the EGFR and open novel possibilities to control EGFR signaling for cancer treatment.

A Constitutive EGFR Kinase Dimer to Study Inhibitor Pharmacology.

Lung cancer is commonly caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric kinase inhibitors are unaffected by common ATP-site resistance mutations and represent a promising therapeutic strategy for targeting drug-resistant EGFR variants. However, allosteric inhibitors are antagonized by kinase dimerization, and understanding this phenomenon has been limited to cellular experiments. To facilitate the study of allosteric inhibitor pharmacology, we designed and purified a constitutive EGFR kinase dimer harboring the clinically relevant L858R/T790M mutations. Kinetic characterization revealed that the EGFR kinase dimer is more active than monomeric EGFR(L858R/T790M) kinase and has the same Km,ATP Biochemical profiling of a large panel of ATP-competitive and allosteric EGFR inhibitors showed that allosteric inhibitor potency decreased by >500-fold in the kinase dimer compared with monomer, yielding IC50 values that correlate well with Ba/F3 cellular potencies. Thus, this readily purifiable constitutive asymmetric EGFR kinase dimer represents an attractive tool for biochemical evaluation of EGFR inhibitor pharmacology, in particular for allosteric inhibitors. SIGNIFICANCE STATEMENT: Drugs targeting epidermal growth factor receptor (EGFR) kinase are commonly used to treat lung cancers but are affected by receptor dimerization. Here, we describe a locked kinase dimer that can be used to study EGFR inhibitor pharmacology.

Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin

Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin, which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of Na+ pump rhodopsin from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.

Coupling and thermosplasmonic properties of asymmetric hexagonal nanoframes

The interaction of light with asymmetric nanostructures leads to the coupling of plasmon modes. Understanding the nature and tunability of these hybridized plasmon modes is essential for both fundamental studies and the development of new applications. Herein, we systematically examine these coupled plasmon modes and heat generated by the asymmetric hexagonal nanoframes dimers (HNDs). Our results show that there are three plasmon bands in the examined wavelength. One of these bands is related to the coupled plasmon modes, which can be actively controlled by changing the height of nanoparticles. These coupled plasmon modes originate from the interaction of dipoles with a minimum splitting energy of 170 meV. Moreover, the thermoplasmonic studies show that the temperature of the asymmetric HNDs strongly depends on the coupling of nanoframes which shows great promise for highly sensitive thermal imaging.

An Influenza A virus can evolve to use human ANP32E through altering polymerase dimerization

Human ANP32A and ANP32B are essential but redundant host factors for influenza virus genome replication. While most influenza viruses cannot replicate in edited human cells lacking both ANP32A and ANP32B, some strains exhibit limited growth. Here, we experimentally evolve such an influenza A virus in these edited cells and unexpectedly, after 2 passages, we observe robust viral growth. We find two mutations in different subunits of the influenza polymerase that enable the mutant virus to use a novel host factor, ANP32E, an alternative family member, which is unable to support the wild type polymerase. Both mutations reside in the symmetric dimer interface between two polymerase complexes and reduce polymerase dimerization. These mutations have previously been identified as adapting influenza viruses to mice. Indeed, the evolved virus gains the ability to use suboptimal mouse ANP32 proteins and becomes more virulent in mice. We identify further mutations in the symmetric dimer interface which we predict allow influenza to adapt to use suboptimal ANP32 proteins through a similar mechanism. Overall, our results suggest a balance between asymmetric and symmetric dimers of influenza virus polymerase that is influenced by the interaction between polymerase and ANP32 host proteins.

Microrobots powered by concentration polarization electrophoresis (CPEP)

Second-order electrokinetic flow around colloidal particles caused by concentration polarization electro-osmosis (CPEO) can result in a phoretic motion of asymmetric particle dimers in a homogeneous AC electrical field, which we refer to as concentration polarization electro-phoresis (CPEP). To demonstrate this actuation mechanism, we created particle dimers from micron-sized silica spheres with sizes 1.0 μm and 2.1 μm by connecting them with DNA linker molecules. The dimers can be steered along arbitrarily chosen paths within a 2D plane by controlling the orientation of the AC electric field in a fluidic chamber with the joystick of a gamepad. Further utilizing induced dipole-dipole interactions, we demonstrate that particle dimers can be used to controllably pick up monomeric particles and release them at any desired position, and also to assemble several particles into groups. Systematic experiments exploring the dependence of the dimer migration speed on the electric field strength, frequency, and buffer composition align with the theoretical framework of CPEO and provide parameter ranges for the operation of our microrobots. Furthermore, experiments with a variety of asymmetric particles, such as fragmented ceramic, borosilicate glass, acrylic glass, agarose gel, and ground coffee particles, as well as yeast cells, demonstrate that CPEP is a generic phenomenon that can be expected for all charged dielectric particles.

THU048 Determination Of Receptor Binding Affinity Of Growth Hormone Analogues To The Growth Hormone Receptor In A Drug Screening Context

Abstract Disclosure: S. Erlendsson: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. A. Kulakova: Employee; Self; Novo Nordisk. S. Poulsen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. P. Thygesen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. Growth hormone (GH) and GH analogues are characterized by their ability to bind and activate the GH receptor (GHR). Binding affinity and functional activity are pivotal parameters for development and characterization of new GH analogues for treatment of growth disorders. The GHR is a member of the class I cytokine family and obtains a tripartite structure consisting of two extracellular fibronectin type III domains (ECD), a single-pass transmembrane domain (TMD), and a disordered intracellular domain (ICD). These receptors lack intrinsic kinase activity but rely on recruitment of intracellular signalling kinases and regulatory proteins. Activation of the downstream JAK-STAT signalling cascade is initiated by formation of an asymmetric GHR dimer induced by binding of GH to the GHR ECDs via two partially overlapping binding sites designated site I and II. The structural rearrangement in the ECDs propagates through the TMD and results in a separation of the ICDs which leads to activation through cross-phosphorylation of the Janus kinases 2 (JAK2). Here we utilize isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and small angle x-ray scattering (SAXS) to characterize the binding of native GH and GH analogues including somapacitan and pegvisomant to the monomeric GHR as well as an artificial soluble stabilized dimeric GHR. The GH analogues with good binding properties were subsequently tested in vitro for biological activity using a STAT3-luciferase reporter gene assay (RGA) and a proliferation assay with BAF3 cells expressing the human GHR. Both assays were established in two formats enabling testing of both receptor agonists and antagonists. Using SPR we show that the binding of all tested compounds is preserved between the monomeric and dimeric GHR with affinities ranging from near-picomolar to lower micromolar and we are subsequently able to confirm the expected stoichiometry by utilizing a combination of ITC and SAXS. In the RGA and the proliferation assay we demonstrate that a 1:2 stoichiometry is critical for receptor activation regardless of the affinity and therefore we propose high throughput screening of receptor stoichiometry as key in discovery of new growth disorder therapeutics. Presentation: Thursday, June 15, 2023

Exact Excited-State Functionals of the Asymmetric Hubbard Dimer.

The exact functionals associated with the (singlet) ground state and the two singlet excited states of the asymmetric Hubbard dimer at half-filling are calculated using both Levy's constrained search and Lieb's convex formulation. While the ground-state functional is, as is commonly known, a convex function with respect to the density, the functional associated with the doubly excited state is found to be concave. Also, because the density-potential mapping associated with the first excited state is noninvertible, its "functional" is a partial, multivalued function composed of one concave and one convex branch that correspond to two separate domains of the external potential. Remarkably, it is found that, although the one-to-one mapping between density and external potential may not apply (as in the case of the first excited state), each state-specific energy and corresponding universal functional are "functions" whose derivatives are each other's inverse, just as in the ground state formalism.

Symmetrical anisotropy enables dynamic diffraction control in photonics.

Despite the steady advancements in nanofabrication made over the past decade that had prompted a plethora of intriguing applications across various fields, achieving compatibility between miniaturized photonic devices and electronic dimensions remains unachievable due to the inherent diffraction limit of photonic devices. Herein, we present an approach based on anisotropic scaling of the shapes of photonic crystals (PhCs) to overcome the diffraction limit and achieve controlled diffraction limit along the ΓX direction. Thus, we demonstrate that scaling the direction perpendicular to the wave's propagation (y-direction) by 1/2 and 1/4 significantly improves the diffraction limit by two and four orders of magnitude, respectively. This approach opens up possibilities for high-frequency wave guiding in a cermet configuration, which was previously unachievable. Furthermore, we illustrate the existence of a quasi-bound state in the continuum (QBICs) in asymmetric dimer network-type photonic crystals (PhCs).

Møller-Plesset and Density-Fixed Adiabatic Connections for a Model Diatomic System at Different Correlation Regimes.

In recent years, adiabatic connection (AC) interpolations developed within density functional theory (DFT) have been found to provide good performances in the calculation of interaction energies when used with Hartree-Fock (HF) ingredients. The physical and mathematical reasons for such unanticipated performance have been clarified, to some extent, by studying the strong-interaction limit of the Møller-Plesset (MP) AC. In this work, we calculate both the MP and the DFT AC integrand for the asymmetric Hubbard dimer, which allows for a systematic investigation of different correlation regimes by varying two simple parameters in the Hamiltonian: the external potential, Δv, and the interaction strength, U. Notably, we find that, while the DFT AC integrand appears to be convex in the full parameter space, the MP integrand may change curvature twice. Furthermore, we discuss different aspects of the second-order expansion of the correlation energy in each AC, and we demonstrate why the derivative of the λ-dependent density in the MP AC at λ = 0 (i.e., at the HF density) is zero in the model. Concerning the strong-interaction limit of both ACs in the Hubbard dimer setting, we show that the asymptotic value of the MP AC, W∞HF, is lower than (or equal to) its DFT analogue, W∞KS, if the two are compared at a given density, just like in real space. However, we also show that this is not always the case if the two quantities are compared at a given external potential.

Proton-decoupled deuterium NMR study of an asymmetric liquid crystal dimer having two nematic phases.

Proton-decoupled deuterium NMR spectra were obtained for an asymmetric liquid crystal dimer 1-(4-cyanobiphenyl-4'-yloxy)-6-(4-cyanobiphenyl-4'-yl)hexane (CB6OCB) containing a single -CD_{2}- group. The sample has two nematic liquid crystal phases: a twist-bend nematic, N_{TB}, at the lowest temperature followed by a uniaxial nematic, N_{U}, on increasing the temperature. Proton decoupling reduces the linewidths of the peaks in the deuterium spectrum from kHz to ∼100Hz, enabling quadrupolar splittings, Δν, to be obtained with enhanced precision as well as the dipolar coupling between deuterium nuclei within the CD_{2} group, hence enhancing the information content.

Symmetry-breaking charge-transfer enables turn-on fluorescence sensing in the shortwave infrared spectral region for in vivo vasculature redox homeostasis

Functional in vivo imaging in the short-wavelength infrared spectral region (SWIR) has broad potential in disease diagnosis, fluorescence guided surgery and in vivo pharmacology. In recent years, dyes absorbing in the SWIR region started to emerge. Yet, probe design based on these SWIR dyes remains difficult due to scarcity of efficient fluorescence quenching mechanism. Here, we propose that symmetry breaking charge transfer is a viable approach to develop off-on type probes with SWIR fluorophores. We synthesized the first V-shaped asymmetric cyanine dimer (VAD1080) and verified that its fluorescence was efficiently quenched. The subsequent treatment with highly oxidative species including ONOO- could be employed to inhibit this photophysical quenching mechanism and restore the fluorescence. Its fluorescence sensing capability of oxidative stress in vivo was exemplified. We discovered that overdose of APAP induced a rapid rise of oxidative stress in the vasculature prior to a prior in the liver. This yields insights over the toxicology of APAP and suggested that clearance of reactive species in blood vessels is be a viable approach as a therapeutic measure of acute APAP toxicity.