Trimeric Structure Research Articles

35B5 antibody potently neutralizes SARS-CoV-2 Omicron by disrupting the N-glycan switch via a conserved spike epitope

The SARS-CoV-2 Omicron variant harbors more than 30 mutations in the spike protein, leading to immune evasion from many therapeutic neutralizing antibodies. We reveal that a receptor-binding domain (RBD)-targeting monoclonal antibody, 35B5, exhibits potent neutralizing efficacy to Omicron. Cryo-electron microscopy structures of the extracellular domain trimer of Omicron spike with 35B5 Fab reveal that Omicron spike exhibits tight trimeric packing and high thermostability, as well as significant antigenic shifts and structural changes, within the RBD, N-terminal domain (NTD), and subdomains 1 and 2. However, these changes do not affect targeting of the invariant 35B5 epitope. 35B5 potently neutralizes SARS-CoV-2 Omicron and other variants by causing significant conformational changes within a conserved N-glycan switch that controls the transition of RBD from the “down” state to the “up” state, which allows recognition of the host entry receptor ACE2. This mode of action and potent neutralizing capacity of 35B5 indicate its potential therapeutic application for SARS-CoV-2.

PotN represents a novel energy-state sensing PII subfamily, occurring in firmicutes.

PII proteins are signal processor proteins that regulate the cellular metabolism of Bacteria, Archea and plant chloroplasts typically in response to the cellular nitrogen status. Here, we report the first biochemical characterization of a novel PII-like protein PotN from Lentilactobacillus hilgardii. PotN is encoded in an operon together with the potABCD genes, encoding the ABC transporter for spermidine/putrescine. Like canonical PII proteins, the native PotN has a trimeric structure and competitively binds ATP and ADP, but it does not bind 2-oxoglutarate. Immunoprecipitation and pull-down experiments revealed that PotN is associated in vivo with the transcriptional regulator GlnR and the beta-subunit of pyruvate/2-oxoglutarate/acetoin dehydrogenase AcoB. Moreover, in vitro assays revealed that the ATPase domain of PotA also is able to interact with PotN. Interaction analyses demonstrated that PotN preferentially associates with PotA in the ADP state, whereas it binds to GlnR at elevated ATP levels. This suggests that PotN regulates the transport of polyamines and GlnR-dependent gene expression in response to the energy availability for the cell.

The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

Structures of the Omicron spike trimer with ACE2 and an anti-Omicron antibody.

The SARS-CoV-2 Omicron variant has become the dominant infective strain. We report the structures of the Omicron spike trimer on its own or in complex with ACE2 or an anti-Omicron antibody. Most Omicron mutations are located on the surface of the spike protein, which change binding epitopes to many current antibodies. In the ACE2 binding site, compensating mutations strengthen RBD binding to ACE2. Both the RBD and the apo form of the Omicron spike trimer are thermodynamically unstable. An unusual RBD-RBD interaction in the ACE2-spike complex supports the open conformation and further reinforces ACE2 binding to the spike trimer. A broad-spectrum therapeutic antibody, JMB2002, which has completed a Phase 1 clinical trial, maintains neutralizing activity against Omicron. JMB2002 binds to RBD differently from other characterized antibodies and inhibits ACE2 binding.

Visualizing Electric and Magnetic Field Coupling in Au-Nanorod Trimer Structures via Stimulated Electron Energy Gain and Cathodoluminescence Spectroscopy: Implications for Meta-Atom Imaging

Visualizing Electric and Magnetic Field Coupling in Au-Nanorod Trimer Structures via Stimulated Electron Energy Gain and Cathodoluminescence Spectroscopy: Implications for Meta-Atom Imaging

Light-Harvesting Complex II Adopts Different Quaternary Structures in Solution as Observed Using Small-Angle Scattering

The high-resolution crystal structure of the trimeric major light-harvesting complex of photosystem II (LHCII) is often perceived as the basis for understanding its light-harvesting and photoprotective functions. However, the LHCII solution structure and its oligomerization or aggregation state may generally differ from the crystal structure and, moreover, also depend on its functional state. In this regard, small-angle scattering experiments provide the missing link by offering structural information in aqueous solution at physiological temperatures. Herein, we use small-angle scattering to investigate the solution structures of two different preparations of solubilized LHCII employing the nonionic detergents n-octyl-β-d-glucoside (OG) and n-dodecyl-β-D-maltoside (β-DM). The data reveal that the LHCII-OG complex is equivalent to the trimeric crystal structure. Remarkably, however, we observe─for the first time─a stable oligomer composed of three LHCII trimers in the case of the LHCII-β-DM preparation, implying additional pigment-pigment interactions. The latter complex is assumed to mimic trimer-trimer interactions which play an important role in the context of photoprotective nonphotochemical quenching.

Clustering of auro-acetylenes via C-Au⋯π interactions: Gold-hydrogen analogy

The structure, stability, electronic and thermochemical properties of small clusters of auro-acetylenes (C2Au2) are investigated using dispersion-corrected density functional theoretical (DFT) method. The studies showed that auro-acetylene clusters are connected mainly via C-Au⋯π interactions analogous to C-H⋯π interactions in acetylene clusters. In the most stable structures of trimer and tetramer, each of the constituent unit acts both as a C-Au⋯π donor and as a C-Au⋯π acceptor forming cyclic structures akin to acetylene clusters. The studies also revealed that the C-Au⋯π interactions are stronger than the aurophilic interactions forming the clusters.

Effect of plasmonic coupling in different assembly of gold nanorods studied by FDTD

The influence of the orientation of gold nanorods in different assemblies has been investigated using the Finite Difference Time Domain (FDTD) simulation method. To understand the relative orientation, we vary the size and angle in dimer geometries. Significant effects of plasmon coupling emerged in longitudinal resonances having end-to-end configurations of gold nanorods. The effect of orientational plasmon coupling in dimers gives rise to both bonding and anti-bonding plasmon modes. Effects of various geometries like primary monomer, dimer, trimer, and tetramer structures have been explored and compared with their higher nanorod ensembles. The asymmetric spectral response in a 4 * 4 gold nanorods array indicates a Fano-like resonance. The variation of gap distance in ordered arrays allowed modulation of the Fano resonance mode. The plasmon modes' resonance wavelength and field enhancement have been tuned by varying the gap distance, angular orientation, size irregularity between the nanorods, and nanorod numbers in an array. The integrated nanostructures studied here are not only significant for fundamental research but also applications in plasmon-based devices.

Co-administration of rotavirus nanospheres VP6 and NSP4 proteins enhanced the anti-NSP4 humoral responses in immunized mice

Inconveniences associated with the efficacy and safety of the World Health Organization (WHO) approved/prequalified live attenuated rotavirus (RV) vaccines, sounded for finding alternative non-replicating modals and proper RV antigens (Ags). Herein, we report the development of a RV candidate vaccine based on the combination of RV VP6 nanospheres (S) and NSP4112-175 proteins (VP6S + NSP4). Self-assembled VP6S protein was produced in insect cells. Analyses by western blotting and transmission electron microscopy (TEM) indicated expression of VP6 trimer structures with sizes of ≥140 kDa and presence of VP6S. Four group of mice were immunized (2-dose formulation) intra-peritoneally (IP) by either¨VP6S + NSP4¨ or each protein alone (VP6S or NSP4112-175) emulsified in aluminium hydroxide or control. Results indicated that VP6S + NSP4 formulation induced significant anti-VP6 IgG (P < 0.001) and IgA (P < 0.05) as well as anti-NSP4 IgG (P < 0.001) and enhancement of protective immunity. Analyses of anti-VP6S and anti-NSP4 IgG subclass (IgG1 and IgG2a) showed IgG1/IgG2a ≥6 and IgG1/IgG2a ≥3 ratios, respectively indicating Th2 polarization of immune responses. The combination of VP6S + NSP4 proteins emulsified in aluminum hydroxide adjuvant might present a dual universal, efficient and cost-effective candidate vaccine against RV infection.

Aggregation Turns BODIPY Fluorophores into Photosensitizers: Reversibly Switching Intersystem Crossing On and Off for Smart Photodynamic Therapy

Aggregation Turns BODIPY Fluorophores into Photosensitizers: Reversibly Switching Intersystem Crossing On and Off for Smart Photodynamic Therapy

Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.

Fractional and composite excitations of antiferromagnetic quantum spin trimer chains

Using quantum Monte Carlo, exact diagonalization, and perturbation theory, we study the spectrum of the S = 1/2 antiferromagnetic Heisenberg trimer chain by varying the ratio g = J2/J1 of the intertrimer and intratrimer coupling strengths. The doublet ground states of trimers form effective interacting S = 1/2 degrees of freedom described by a Heisenberg chain. Therefore, the conventional two-spinon continuum of width ∝ J1 when g = 1 evolves into to a similar continuum of width ∝ J2 when g → 0. The intermediate-energy and high-energy modes are termed doublons and quartons which fractionalize with increasing g to form the conventional spinon continuum. In particular, at g ≈ 0.716, the gap between the low-energy spinon branch and the high-energy band with mixed doublons, quartons, and spinons closes. These features should be observable in inelastic neutron scattering experiments if a quasi-one-dimensional quantum magnet with the linear trimer structure and J2 < J1 can be identified. Our results may open a window for exploring the high-energy fractional excitations.

Structural insights into blue-green light utilization by marine green algal light harvesting complex II at 2.78 Å

Light-harvesting complex II (LHCII) present in plants and green algae absorbs solar energy to promote photochemical reactions. A marine green macroalga, Codium fragile, exhibits the unique characteristic of absorbing blue-green light from the sun during photochemical reactions while being underwater owing to the presence of pigment-altered LHCII called siphonaxanthin–chlorophyll a/b-binding protein (SCP). In this study, we determined the structure of SCP at a resolution of 2.78 Å using cryogenic electron microscopy. SCP has a trimeric structure, wherein each monomer containing two lutein and two chlorophyll a molecules in the plant-type LHCII are replaced by siphonaxanthin and its ester and two chlorophyll b molecules, respectively. Siphonaxanthin occupies the binding site in SCP having a polarity in the trimeric inner core, and exhibits a distorted conjugated chain comprising a carbonyl group hydrogen bonded to a cysteine residue of apoprotein. These features suggest that the siphonaxanthin molecule is responsible for the characteristic green absorption of SCP. The replaced chlorophyll b molecules extend the region of the stromal side chlorophyll b cluster, spanning two adjacent monomers.

The reaction between the bromine atom and the water trimer: high level theoretical studies.

Three different reaction pathways are found for the reaction of bromine atom (Br) with the lowest-energy structure of the water trimer [uud-(H2O)3], initially using the MPW1K-DFT method. The three bromine pathways have closely related geometries and energetics, analogous to those found for the fluorine and chlorine reactions. The lowest-energy pathway of the Br + uud-(H2O)3 reaction was further investigated using the "gold standard" CCSD(T) method and the correlation-consistent basis sets up to cc-pVQZ(-PP). Based on the CCSD(T)/cc-pVQZ(-PP)//CCSD(T)/cc-pVTZ(-PP) results, the Br + (H2O)3 reaction is endothermic by 33.3 kcal mol-1. The classical barrier height is 29.0 kcal mol-1 between the reactants and the exit complex, and there is no barrier for the reverse reaction. The Br⋯(H2O)3 entrance complex is found to lie 4.7 kcal mol-1 below the separated reactants, and the HBr⋯(H2O)2OH exit complex is bound by 6.4 kcal mol-1 relative to the separated products. This potential energy profile is further corrected by the zero point energies and spin-orbit coupling effects. Structurally, the Br + (H2O)3 stationary points can be derived from those of the simpler Br + (H2O)2 reaction by judiciously appending a H2O molecule. The Br + (H2O)3 potential energy profile is compared with the Br + (H2O)2 and Br + H2O reactions, as well as to the valence isoelectronic Cl + (H2O)3 and F + (H2O)3 systems. It is reasonable to expect that the reactions between the bromine atom and larger water clusters would be similar to the Br + (H2O)3 reaction.

Frustration-driven allosteric regulation and signal transmission in the SARS-CoV-2 spike omicron trimer structures: a crosstalk of the omicron mutation sites allosterically regulates tradeoffs of protein stability and conformational adaptability.

Dissecting the regulatory principles underlying function and activity of the SARS-CoV-2 spike protein at the atomic level is of paramount importance for understanding the mechanisms of virus transmissibility and immune escape. In this work, we introduce a hierarchical computational approach for atomistic modeling of allosteric mechanisms in the SARS-CoV-2 Omicron spike proteins and present evidence of a frustration-based allostery as an important energetic driver of the conformational changes and spike activation. By examining conformational landscapes and the residue interaction networks in the SARS-CoV-2 Omicron spike protein structures, we have shown that the Omicron mutational sites are dynamically coupled and form a central engine of the allosterically regulated spike machinery that regulates the balance and tradeoffs between conformational plasticity, protein stability, and functional adaptability. We have found that the Omicron mutational sites at the inter-protomer regions form regulatory hotspot clusters that control functional transitions between the closed and open states. Through perturbation-based modeling of allosteric interaction networks and diffusion analysis of communications in the closed and open spike states, we have quantified the allosterically regulated activation mechanism and uncover specific regulatory roles of the Omicron mutations. Atomistic reconstruction of allosteric communication pathways and kinetic modeling using Markov transient analysis reveal that the Omicron mutations form the inter-protomer electrostatic bridges that operate as a network of coupled regulatory switches that could control global conformational changes and signal transmission in the spike protein. The results of this study have revealed distinct and yet complementary roles of the Omicron mutation sites as a network of hotspots that enable allosteric modulation of structural stability and conformational changes which are central for spike activation and virus transmissibility.

Immunity analysis against Fowl Adenovirus serotype 4 (FAdV-4) based on Fiber-2 trimer Protein with the different virulence

Since June 2015, Fowl adenovirus outbreaks have occurred in China, causing significant economic losses to poultry industry. The FAdV-4 Fiber-2 proteins could induce effective protection, but the precise mechanism of immune protection remains unknown. Here, we have compared the biological characteristics of Fiber-2 protein of the very virulent WZ strain of FAdV-4 (vvFAdV-4) with that of non-virulent ON1 strain. The sequence analysis revealed natural deletions and sequence differences between the classical non-pathogenic strain ON1 and the vvFAdV-4 isolate. These two Fiber-2 proteins successfully expressed in E. coli resemble in structure and function to the native-like trimeric protein. The trimeric structure and bioreactivity of the recombinant Fiber-2 proteins to FAdV-4 specific antibodies were characterized. The immune protection induced by Fiber-2 proteins of FAdV-4 WZ and ON1 strains were compared in SPF chickens. All birds in the WZ-Fiber-2 immunized group generated systemic specific antibodies compared with both ON1-Fiber-2 protein and PBS immunized groups. According to the results of attack mortalities, viral shedding and tissue gross lesion, the WZ Fiber-2 protein induced complete protection at a dose of 2 μg per chicken, whereas the ON1-Fiber-2 protein induced 0 protection at 3 dpc. In view of the characteristics of Fiber-2 proteins of different strains, this study can help us to further understand the mechanism of protective immunity and provide a basis for the prevention and control of FAdV-4 in chickens.

A cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI

Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.

La induced Si3 trimer monolayer on Si(111) surface: an ab initio study

The π-bond Seiwatz chain (SC) is one of the well-known reconstruction induced by alkaline or rare earth metals on Si(111) surface. Here we identify by ab initio calculations a new reconstruction of La induced quasi-two-dimensional Si3 trimer monolayer on Si(111)- surface. Its surface unit cell has one La atom and one Si3 trimer with the same La coverage (1/3 monolayer) as SC structure and the Si3 trimer satisfies the electron counting rule with a transfer of valence electrons from La atom, formally as , in correspondence to the milkstool model for Bi trimers on Si(111) surface. Band structure calculations show a semiconducting character with an indirect surface band gap of 0.76 eV. Moreover, a two-stage conversion process between the Si3 trimer and SC structure is verified by the climbing-image nudged elastic band method. These findings pave the way for further exploration of the new surface structure and its outstanding properties.

Structure, function and pharmacology of human itch receptor complexes.

In the clades of animals that diverged from the bony fish, a group of Mas-related G-protein-coupled receptors (MRGPRs) evolved that have an active role in itch and allergic signals1,2. As an MRGPR, MRGPRX2 is known to sense basic secretagogues(agents that promotesecretion) and is involved in itch signals and eliciting pseudoallergic reactions3-6. MRGPRX2 has been targeted by drug development efforts to prevent the side effects induced by certain drugs or to treat allergic diseases. Here we report a set of cryo-electron microscopy structures of the MRGPRX2-Gi1 trimer in complex with polycationic compound 48/80 or with inflammatory peptides. The structures of the MRGPRX2-Gi1 complex exhibited shallow, solvent-exposed ligand-binding pockets. We identified key common structural features of MRGPRX2 and describe a consensus motif for peptidic allergens. Beneath the ligand-binding pocket, the unusual kink formation at transmembrane domain 6 (TM6) and the replacement of the general toggle switch from Trp6.48 to Gly6.48 (superscript annotations as per Ballesteros-Weinstein nomenclature) suggest a distinct activation process. We characterized the interfaces of MRGPRX2 and the Gi trimer, and mapped the residues associated with key single-nucleotide polymorphisms on both the ligand and G-protein interfaces of MRGPRX2. Collectively, our results provide a structural basis for the sensing of cationic allergens by MRGPRX2, potentially facilitating the rational design of therapies to prevent unwanted pseudoallergic reactions.

Controlled Modular Multivalent Presentation of the CD40 Ligand on P22 Virus-like Particles Leads to Tunable Amplification of CD40 Signaling

Ligands of the tumor necrosis factor superfamily (TNFSF) are appealing targets for immunotherapy research due to their integral involvement in stimulation or restriction of immune responses. TNFSF-targeted therapies are currently being developed to combat immunologically based diseases and cancer. A crucial determinant of effective TNFSF receptor binding and signaling is the trimeric quaternary structure of the ligand. Additionally, ligand multivalency is essential to propagate strong signaling in effector cells. Thus, designing a synthetic platform to display trimeric TNFSF ligands in a multivalent manner is necessary to further the understanding of ligand-receptor interactions. Viral nanocages have architectures that are amenable to genetic and chemical modifications of both their interior and exterior surfaces. Notably, the exterior surface of virus-like particles can be utilized as a platform for the modular multivalent presentation of target proteins. In this study, we build on previous efforts exploring the bacteriophage P22 virus-like particle for the exterior multivalent modular display of a potent immune-stimulating TNFSF protein, CD40 ligand (CD40L). Using a cell-based reporter system, we quantify the effects of tunable avidity on CD40 signaling by CD40L displayed on the surface of P22 nanocages. Multivalent presentation of CD40L resulted in a 53.6-fold decrease of the half maximal effective concentration (EC50) compared to free CD40L, indicating higher potency. Our results emphasize the power of using P22-based biomimetics to study ligand-receptor interactions within their proper structural context, which may contribute to the development of effective immune modulators.