Asymmetric Trimer Research Articles

PtuA and PtuB assemble into an inflammasome-like oligomer for anti-phage defense.

Escherichia coli Septu system, an anti-phage defense system, comprises two components: PtuA and PtuB. PtuA contains an ATPase domain, while PtuB is predicted to function as a nuclease. Here we show that PtuA and PtuB form a stable complex with a 6:2 stoichiometry. Cryo-electron microscopy structure of PtuAB reveals a distinctive horseshoe-like configuration. PtuA adopts a hexameric arrangement, organized as an asymmetric trimer of dimers, contrasting the ring-like structure by other ATPases. Notably, the three pairs of PtuA dimers assume distinct conformations and fulfill unique roles in recruiting PtuB. Our functional assays have further illuminated the importance of the oligomeric assembly of PtuAB in anti-phage defense. Moreover, we have uncovered that ATP molecules can directly bind to PtuA and inhibit the activities of PtuAB. Together, the assembly and function of the Septu system shed light on understanding other ATPase-containing systems in bacterial immunity.

Magnetic behaviour of a spin-canted asymmetric lanthanide quinolate trimer.

An asymmetrical dysprosium trimer with a molecular formula of [Dy3(hq)7(hqH)(NO3)2(H2O)] was obtained through a reflux reaction employing as starting material Dy(NO3)3·nH2O and 8-quinolinoline as ligand. Magnetic susceptibility investigations show the system to be an SMM, which was corroborated by sub-Kelvin μSQUID studies. Upon cooling, the magnetic susceptibility also exhibits a decrease in the χMT product, which was confirmed to be due to intramolecular antiferromagnetic interactions. μSQUID measurements, moreover, reveal a marked magnetic behaviour in the angular dependence of the hysteresis loops. The latter is a direct consequence of the non-colinear spin arrangement of the anisotropy axes of each Dy(III) ion in [Dy3(hq)7(hqH)(NO3)2(H2O)] and the interaction between the ions, as also evidenced by CASSCF calculations. Our results evidence the effect of spin canting along with the intramolecular interactions, which can induce non-trivial magnetic behaviour in SMMs.

Stationary states and dynamic regimes for a nonlinear asymmetric trimer with gain and loss

The paper studies a model of three couplers with Kerr-type nonlinearity in an asymmetric form with gain and loss. Analytical solutions of nonlinear stationary states are constructed in the vicinity of the neutral stability parameters for the null solution. The stability of such states is investigated analytically. Families of nonlinear stationary states depending on the gain coefficient are built numerically. The study of stationary solutions has revealed the parameters of oscillating and quasi-periodic dynamic regimes, which turn out to be attractors for a wide variety of initial data. The investigated properties may be useful for creating optical processing devices.

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.

Crystal structures of multidrug efflux transporters from Burkholderia pseudomallei suggest details of transport mechanism

BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae, where both are symmetric trimers composed of three "binding"-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.

Effective plasmonic spectroscopic amplifiers for hyper-Raman scattering process

In an asymmetric Au cubic trimer, influence of the rotation angle (θ) and side length (w) on both plasmonic coupling features and corresponding enhancement factor of hyper-Raman scattering (HRS) process have been investigated comprehensively under the illumination of a longitudinally polarized light. The finite-difference time-domain (FDTD) electrodynamic simulation tool has been employed to calculate the optical cross-section and associated nearfield intensity of the irradiated coupled resonators. As θ increases, the polarization state that dominates the coupling phenomenon is gradually switched from facing sides into facing edges which results in (1) a dramatic change in the spectral response of the trimer and (2) a significant improvement in the nearfield intensity that is directly related to the improvement of HRS signal. Breaking size symmetry of the cubic trimer provides a novel approach to reach the desired spectral response that permits such trimer to be used as an active substrate for HRS procedures. After optimizing both the orientation angle and size of the interacting plasmonic characters forming of the trimer, the enhancement factor of HRS process can reach a value never reported before as high as 1 × 1021.

Articles of Significant Interest in This Issue

The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by cleavage of the gp160 precursor. Zhang et al. (e00529-21) find that this Env precursor is conformationally flexible and that this plasticity contributes to the efficiency of cleavage. Cryo-electron microscopy (cryo-EM) structures of the uncleaved Env reveal two asymmetric trimer conformations that potentially allow exposure of epitopes for weakly neutralizing antibodies. Selective pressure to maintain flexibility in the precursor of the functional Env allows the uncleaved Env trimer to sample asymmetric conformations that can misdirect host antibody responses toward ineffective neutralizers.

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.

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer–small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF–TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein–protein interactions.

Structural and functional characterization of the deep-sea thermophilic bacteriophage GVE2 tailspike protein

The genome of the thermophilic bacteriophage GVE2 encodes a putative tailspike protein (GVE2 TSP). Here we report the crystal structure of the truncated GVE2 TSP at 2.0-Å resolution lacking 204 amino acid residues at its N-terminus (ΔnGVE2 TSP), possessing a “vase” outline similar to other TSP's structures. However, ΔnGVE2 TSP displays structural characteristics distinct from other TSPs. Despite lacking 204 amino acid residues, the head domain forms an asymmetric trimer compared to symmetric in other TSPs, suggesting that its long N-terminus may be unique to the long-tailed bacteriophages. Furthermore, the α-helix of the neck is 5–7 amino acids longer than that of other TSPs. The most striking feature is that its binding domain consists of a β-helix with 10 turns, whereas other TSPs have 13 turns, even including the phage Sf6 TSP, which is the closest homologue of GVE2 TSP. The C-terminal structure is also quite different with those of other TSPs. Furthermore, we observed that ΔnGVE2 TSP can slow down growth of its host, demonstrating that this TSP is essential for the phage GVE2 to infect its host. Overall, the structural characteristics suggest that GVE2 TSP may be more primitive than other phage TSPs.

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.

Single-Molecule FRET Delineates Asymmetric Trimer Conformations during HIV-1 Entry

HIV-1 entry into cells requires binding of the viral envelope glycoprotein (Env) to receptor CD4 and coreceptor. Imaging of individual Env molecules on native virions revealed that Env trimers are dynamic, spontaneously transitioning between three distinct conformations. Binding of CD4 and coreceptor surrogate antibody 17b promotes opening of the closed Env (State 1) to stabilize an activated conformation (State 3) by way of at least one structural intermediate (State 2). Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we identify this intermediate as an asymmetric conformation where only a single CD4 molecule engages the Env trimer and individual protomers adopt distinct conformations. Binding of a second CD4 molecule is needed to completely open the trimer. Thus, our work reveals a novel asymmetric structural intermediate and gives insights into cooperativity during the opening of the HIV-1 Env trimer.

How do Glutamate Transporters Function as Transporters and Ion Channels?

Plasma-membrane glutamate transporters move the main excitatory neurotransmitter in the mammalian brain, glutamate, across the membrane against its concentration gradient. This uphill transport is achieved by coupling transmembrane glutamate movement to the cotransport of three Na+ ions down their own concentration gradient. In addition, one potassium ion is countertransported.

The structure of the asymmetric helium trimer (3)He(4)He2.

Despite their apparent simplicity, the properties of the two helium trimers, (4)He3 and (3)He(4)He2, are still not completely understood. In particular, the existence of a bound state of the asymmetric trimer (3)He(4)He2 was established many years ago, using different theoretical approaches, and later it was experimentally detected. However its structural properties have not been thoroughly investigated so far, probably because an accurate theoretical description of this very weakly bound system is computationally quite demanding. In this work we give for the first time an accurate and complete theoretical description of the geometrical structure of this fragile system using quantum Monte Carlo techniques employing the TTY potential and compare its properties with those of (4)He2 and (4)He3. We compute average values of distances and angles, along with the angle-angle distribution function: a two-dimensional probability distribution well suited to discuss the shape of a trimer. Our analysis shows that the lighter isotope is very diffuse and can be found at large distances from the other two atoms, but also close to the center of mass of the system in nearly linear configurations. For this system the concept of "equilibrium structure" is meaningless and all kinds of three-atom configurations must be taken into account in its description.

Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog

Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na(+)-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, Glt(Ph), suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3-4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, Glt(Ph) is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved Glt(Ph) retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process.

The Periplasmic Loop Provides Stability to the Open State of the CorA Magnesium Channel

Crystal structures of the CorA Mg(2+) channel have suggested that metal binding in the cytoplasmic domain stabilizes the pentamer in a closed conformation. The open "metal free" state of the channel is, however, still structurally uncharacterized. Here, we have attempted to map conformational states of CorA from Thermotoga maritima by determining which residues support the pentameric structure in the presence or absence of Mg(2+). We find that when Mg(2+) is present, the pentamer is stabilized by the putative gating sites (M1/M2) in the cytoplasmic domain. Strikingly however, we find that the conserved and functionally important periplasmic loop is vital for the integrity of the pentamer when Mg(2+) is absent from the M1/M2 sites. Thus, although the periplasmic loops were largely disordered in the x-ray structures of the closed channel, our data suggests a prominent role for the loops in stabilizing the open conformation of the CorA channels.

Mimicking trimeric interactions in the aromatic side chains of the proteins: A gas phase study of indole…(pyrrole)2 heterotrimer

Aromatic trimeric interactions are extremely significant in the stabilization of the specific structures of the proteins as well as protein-protein, and protein-ligand interactions. Here we have reported a direct evidence of the observation of a cyclic asymmetric structure of indole...(pyrrole)(2) trimer bound by three N-H...π hydrogen bonding interactions in a supersonic jet. The experiment has been performed by using resonant two-photon ionization (R2PI), IR-UV, and UV-UV double resonance spectroscopic techniques. Density functional theory (DFT) calculations nicely corroborate the experimental results showing one weakly allowed IR-active band due to symmetric stretch of the N-H bonds and two strongly allowed IR-active bands due to two types of asymmetric stretches of the N-H bonds in the trimer. The present spectroscopic investigation demonstrates that the strength of the three N-H...π bound intermolecular interactions in the cyclic asymmetric trimer is quite different unlike the corresponding interactions of similar strength in a cyclic symmetric trimer.

Crystal structure of an asymmetric trimer of a bacterial glutamate transporter homolog

We report a structure of a trimeric glutamate transporter homolog from Pyrococcus horikoshii with two protomers in an inward facing state and the third in an intermediate conformation between the outward and inward facing states. The intermediate shows a cavity in the thinnest region of the transporter, which is potentially accessible to extracellular and cytoplasmic solutions. Our findings suggest a structural principle by which transport intermediates may mediate uncoupled permeation of polar solutes.

Crystal Structure of an Asymmetric Trimer of a Bacterial Glutamate Transporter Homologue

Excitatory amino acid transporters (EAATs), or glutamate transporters, are trimeric electrochemically driven pumps. In the brain, EAATs reside in membranes of glial cells and neurons, and couple the uptake of the neurotransmitter glutamate to the co-transport of three sodium ions and a proton, and to the counter-transport of a potassium ion. Also, they mediate a bidirectional chloride flux, which is gated by glutamate and sodium binding but is not required for transport. This channel-like activity may play an important role in regulating synaptic transmission, particularly in retina. Insights into EAATs transport mechanism came from crystal structures of an archaeal homolog, GltPh, which pictured symmetrical trimers with protomers in either outward or inward facing conformations. These structures show that each protomer harbors a distinct transport domain, which coordinates substrate and coupled ions, and traverses ∼15 Å of the lipid bilayer to move its cargo between extracellular and intracellular solutions. Here, we report an asymmetric structure of GltPh with two protomers in the inward facing conformation and one protomer in a novel state, which may represent an early intermediate between outward and inward facing states. This structure illustrates the autonomous nature of the transport domain, consistent with the functional independence of the protomers. It further suggests that transitions between outward and inward facing states may proceed via relatively low-energy intermediates, and provides a glimpse of their unusual structural properties. Specifically, the intermediate state shows compromised packing between protein domains that results in the opening of a lipid-exposed crevice and the formation of a cavity, potentially accessible to the solvent. Based on this structure and published electrophysiology data, we hypothesize that transport cycle intermediates may serve as conduits for uncoupled permeation by polar molecules and ions. Further experiments will be presented.

Elastic network model-based normal mode analysis reveals the conformational couplings in the tripartite AcrAB-TolC multidrug efflux complex

The AcrAB-TolC drug efflux system, energized by proton movement down the transmembrane electrochemical gradient, is responsible for the resistance of the organism to a wide range of drugs. Experimental data suggest functional roles of each part of the assembly, but the detailed working mechanism of this machinery remains elusive. We used elastic network-based normal mode analysis (NMA) to explore the conformational dynamics of the AcrAB-TolC complex. The intrinsic flexibilities of the pore domain in AcrB monomer conform to the previously proposed three-step functionally rotating mechanism for asymmetric AcrB trimer. Conformational couplings across monomers in the AcrB trimer were observed, and the coupling between the transmembrane domain and the other parts of AcrB are strengthened through trimeric assembly. In the tripartite AcrAB-TolC assembly obtained through molecular docking, concerted motions were observed not only at the direct contact interfaces between various components but also between distant parts of the whole complex. The presence of AcrA was shown to significantly strengthen the motional couplings between AcrB and TolC. Overall, NMA revealed an allosteric network in the AcAB-TolC efflux system, which provides hints to our understanding of its detailed working mechanism.