Dimer Formation Research Articles

Structural insights into the regulation, ligand recognition, and oligomerization of bacterial STING

The cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) signaling pathway plays a critical protective role against viral infections. Metazoan STING undergoes multilayers of regulation to ensure specific signal transduction. However, the mechanisms underlying the regulation of bacterial STING remain unclear. In this study, we determined the crystal structure of anti-parallel dimeric form of bacterial STING, which keeps itself in an inactive state by preventing cyclic dinucleotides access. Conformational transition between inactive and active states of bacterial STINGs provides an on-off switch for downstream signaling. Some bacterial STINGs living in extreme environment contain an insertion sequence, which we show codes for an additional long lid that covers the ligand-binding pocket. This lid helps regulate anti-phage activities. Furthermore, bacterial STING can bind cyclic di-AMP in a triangle-shaped conformation via a more compact ligand-binding pocket, forming spiral-shaped protofibrils and higher-order fibril filaments. Based on the differences between cyclic-dinucleotide recognition, oligomerization, and downstream activation of different bacterial STINGs, we proposed a model to explain structure-function evolution of bacterial STINGs.

Exploring protein-protein interactions and oligomerization state of pulmonary surfactant protein C (SP-C) through FRET and fluorescence self-quenching.

Pulmonary surfactant (PS) is a lipid-protein complex that forms films reducing surface tension at the alveolar air-liquid interface. Surfactant protein C (SP-C) plays a key role in rearranging the lipids at the PS surface layers during breathing. The N-terminal segment of SP-C, a lipopeptide of 35 amino acids, contains two palmitoylated cysteines, which affect the stability and structure of the molecule. The C-terminal region comprises a transmembrane α-helix that contains a ALLMG motif, supposedly analogous to a well-studied dimerization motif in glycophorin A. Previous studies have demonstrated the potential interaction between SP-C molecules using approaches such as Bimolecular Complementation assays or computational simulations. In this work, the oligomerization state of SP-C in membrane systems has been studied using fluorescence spectroscopy techniques. We have performed self-quenching and FRET assays to analyze dimerization of native palmitoylated SP-C and a non-palmitoylated recombinant version of SP-C (rSP-C) using fluorescently labeled versions of either protein reconstituted in different lipid systems mimicking pulmonary surfactant environments. Our results reveal that doubly palmitoylated native SP-C remains primarily monomeric. In contrast, non-palmitoylated recombinant SP-C exhibits dimerization, potentiated at high concentrations, especially in membranes with lipid phase separation. Therefore, palmitoylation could play a crucial role in stabilizing the monomeric α-helical conformation of SP-C. Depalmitoylation, high protein densities as a consequence of membrane compartmentalization, and other factors may all lead to the formation of protein dimers and higher-order oligomers, which could have functional implications under certain pathological conditions and contribute to membrane transformations associated with surfactant metabolism and alveolar homeostasis.

DFT investigation of the regioselective allylation of pyrimidine 2′-deoxynucleosides

To understand the regioselectivity observed in the allylation of pyrimidine nucleosides and to identify the factors directing the reaction, a theoretical study of the regioselective allylation was carried out. Several key points were considered such as: the structure of the deprotonated nucleobase in the presence of Na+; the effect of the solvent on the dissociation and aggregation reactions of thymidine/Na+ ion pair; and the likely allylation reaction mechanisms involved. The results showed that the regioselectivity observed experimentally can be attributed to a greater stability of a dimeric form coupled to an increase of the reaction barrier in THF due to larger Na+ binding to the nucleobase.

A Model of the Full-Length Cytokinin Receptor: New Insights and Prospects.

Cytokinins (CK) are one of the most important classes of phytohormones that regulate a wide range of processes in plants. A CK receptor, a sensor hybrid histidine kinase, was discovered more than 20 years ago, but the structural basis for its signaling is still a challenge for plant biologists. To date, only two fragments of the CK receptor structure, the sensory module and the receiver domain, were experimentally resolved. Some other regions were built up by molecular modeling based on structures of proteins homologous to CK receptors. However, in the long term, these data have proven insufficient for solving the structure of the full-sized CK receptor. The functional unit of CK receptor is the receptor dimer. In this article, a molecular structure of the dimeric form of the full-length CK receptor based on AlphaFold Multimer and ColabFold modeling is presented for the first time. Structural changes of the receptor upon interacting with phosphotransfer protein are visualized. According to mathematical simulation and available data, both types of dimeric receptor complexes with hormones, either half- or fully liganded, appear to be active in triggering signals. In addition, the prospects of using this and similar models to address remaining fundamental problems of CK signaling were outlined.

The Impact of PSR™ (Plant Small RNA Technology), Tea Extract, and Its Principal Components on Mitochondrial Function and Antioxidant Properties in Skin Cells

Objective: This study explored the impact of a black tea extract obtained through (plant small RNA) PSRTM technology, characterized by its abundance of small molecules, particularly citric acid—an antioxidant and tricarboxylic acid (TCA) cycle contributor—on mitochondrial health. The primary focus was to assess whether this extract could counteract reactive oxygen species (ROS)-induced mitochondrial alterations associated with aging, which lead to impaired mitochondrial function, reduced ATP production, and increased ROS generation. Methods: The PSRTM extraction method was employed to obtain a high content of polyphenols and small molecules, particularly citric acid. Results: In comparison with a conventional extract, the PSRTM extract demonstrated significant enhancements in aconitase activity, an ROS-sensitive enzyme in the TCA cycle, as well as basal respiration and ATP synthesis in fibroblast cells and skin biopsies. Moreover, the PSRTM extract effectively reduced ROS production by safeguarding this critical enzyme within the Krebs cycle and displayed superior capabilities in scavenging free radicals when exposed to UV-induced stress. When administered post-UV exposure, the PSRTM extract protected nuclear DNA by reducing the formation of cyclobutane pyrimidine dimers (CPDs) and promoting DNA repair mechanisms. Furthermore, the extract exhibited beneficial effects on the extracellular matrix, characterized by a reduction in matrix metalloprotease 1 (MMP1) and an increase in fibrillin 1 expression. Conclusions: These findings collectively suggest that the PSRTM extract holds promising antiaging potential, potentially functioning as a mitochondrial nutrient/protector due to its multifaceted benefits on mitochondrial function, nuclear DNA integrity, and the extracellular matrix.

Dimeric ACE2-FC is equivalent to monomeric ACE2 in the surrogate virus neutralization test

Angiotensin converting enzyme 2 (ACE2) is the main cellular receptor for the dangerous sarbecoviruses SARS-CoV and SARS-CoV-2. Its recombinant extracellular domain is used to monitor the level of the protective humoral immune response to a viral infection or vaccine using a surrogate virus neutralization test (sVNT). Soluble ACE2 is also being considered as an antiviral therapy option potentially insensitive to changes in the SARS-CoV-2 spike protein. For widespread sVHT testing, it is necessary to use ACE2 preparations or ACE2 conjugates with constant properties. Previously, we obtained a cell line that produces soluble monomeric ACE2 and showed that this variant of ACE2 can be used in sBHT, preferably in the form of a conjugate with horseradish peroxidase. To obtain a stable and universally applicable form of soluble ACE2, a cell line was obtained that produced the ACE2-Fc fusion protein with high productivity, more than 150 mg/l of the target protein during cultivation in a stirred flask. Affinity-purified ACE2-Fc is a mixture of dimeric and tetrameric forms, but allows one to obtain linearizable antibody inhibition curves for complexation with the receptor-binding domain of the SARS-CoV-2 spike protein. The ACE2-Fc-HRP based sVHT testing system can be used to practically measure the levels of virus-neutralizing antibodies against various circulating variants of the SARS-CoV-2 virus.

A Rigorous Framework for Calculating Protein-Protein Binding Affinities in Membranes.

Calculating the binding free energy of integral transmembrane (TM) proteins is crucial for understanding the mechanisms by which they recognize one another and reversibly associate. The glycophorin A (GpA) homodimer, composed of two α-helical segments, has long served as a model system for studying TM protein reversible association. The present work establishes a methodological framework for calculating the binding affinity of the GpA homodimer in the heterogeneous environment of a membrane. Our investigation carefully considered a variety of protocols, including the appropriate choice of the force field, rigorous standardization reflecting the experimental conditions, sampling algorithm, anisotropic environment, and collective variables, to accurately describe GpA dimerization via molecular dynamics-based approaches. Specifically, two strategies were explored: (i) an unrestrained potential mean force (PMF) calculation, which merely enhances sampling along the separation of the two binding partners without any restraint, and (ii) a so-called "geometrical route", whereby the α-helices are progressively separated with imposed restraints on their orientational, positional, and conformational degrees of freedom to accelerate convergence. Our simulations reveal that the simplified, unrestrained PMF approach is inadequate for the description of GpA dimerization. Instead, the geometrical route, tailored specifically to GpA in a membrane environment, yields excellent agreement with experimental data within a reasonable computational time. A dimerization free energy of -10.7 kcal/mol is obtained, in fairly good agreement with available experimental data. The geometrical route further helps elucidate how environmental forces drive association before helical interactions stabilize it. Our simulations also brought to light a distinct, long-lived spatial arrangement that potentially serves as an intermediate state during dimer formation. The methodological advances in the generalized geometrical route provide a powerful tool for accurate and efficient binding-affinity calculations of intricate TM protein complexes in inhomogeneous environments.

Self-Assembly and Conformational Change in the Oligomeric Structure of the Ectodomain of the TBEV E Protein Studied via X-ray, Small-Angle X-ray Scattering, and Molecular Dynamics

The determination of the three-dimensional structures of viral proteins is a necessary step both for understanding the mechanisms of virus pathogenicity and for developing methods to combat viral infections. This study aimed to explore the folding and oligomeric state of the major component of the virion surface of the tick-borne encephalitis virus (TBEV), the ectodomain of the envelope E protein (ectoE), which was expressed in E. coli in a soluble form and purified from inclusion bodies as a mixture of dimeric and monomeric forms. The time-dependent assembly of monomers into dimers was detected using size-exclusion chromatography. An X-ray diffraction study of the ectoE crystals grown at pH 4.5 confirmed the dimeric folding of the recombinant protein typical for ectoE. The ability of ectoE dimers to self-assemble into tetramers was detected via small-angle X-ray scattering (SAXS) in combination with molecular dynamics. Such self-assembly occurred at protein concentrations above 4 mg/mL and depended on the pH of the solution. In contrast to stable, specific dimers, we observed that tetramers were stabilized with weak intermolecular contacts and were sensitive to environmental conditions. We discovered the ability of ectoE tetramers to change conformation under crystallization conditions. These results are important for understanding the crystallization process of viral proteins and may be of interest for the development of virus-like particles.

Elucidating structural variability in p53 conformers using combinatorial refinement strategies and molecular dynamics

ABSTRACT Low molecular weight proteins and protein assemblies can now be investigated using cryo-electron microscopy (EM) as a complement to traditional structural biology techniques. It is important, however, to not lose sight of the dynamic information inherent in macromolecules that give rise to their exquisite functionality. As computational methods continue to advance the field of biomedical imaging, so must strategies to resolve the minute details of disease-related entities. Here, we employed combinatorial modeling approaches to assess flexible properties among low molecular weight proteins (~100 kDa or less). Through a blend of rigid body refinement and simulated annealing, we determined new hidden conformations for wild type p53 monomer and dimer forms. Structures for both states converged to yield new conformers, each revealing good stereochemistry and dynamic information about the protein. Based on these insights, we identified fluid parts of p53 that complement the stable central core of the protein responsible for engaging DNA. Molecular dynamics simulations corroborated the modeling results and helped pinpoint the more flexible residues in wild type p53. Overall, the new computational methods may be used to shed light on other small protein features in a vast ensemble of structural data that cannot be easily delineated by other algorithms.

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.

Substitution of arginine 219 by glycine compromises stability, dimerization, and catalytic activity in a G6PD mutant

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common enzymopathies in humans, present in approximately half a billion people worldwide. More than 230 clinically relevant G6PD mutations of different classes have been reported to date. We hereby describe a patient with chronic hemolysis who presents a substitution of arginine by glycine at position 219 in G6PD protein. The variant was never described in an original publication or characterized on a molecular level. In the present study, we provide structural and biochemical evidence for the molecular basis of its pathogenicity. When compared to the wild-type enzyme, the Arg219Gly mutation markedly reduces the catalytic activity by 50-fold while having a negligible effect on substrate binding affinity. The mutation preserves secondary protein structure, but greatly decreases stability at higher temperatures and to trypsin digestion. Size exclusion chromatography elution profiles show monomeric and dimeric forms for the mutant, but only the latter for the wild-type form, suggesting a critical role of arginine 219 in G6PD dimer formation. Our findings have implications in the development of small molecule activators, with the goal of rescuing the phenotype observed in this and possibly other related mutants.

Each N-glycan on human IgA and J-chain uniquely affects oligomericity and stability

BackgroundImmunoglobulin A (IgA) plays a pivotal role in various immune responses, especially that of mucosal immunity. IgA is usually assembled into dimers with the contribution of J-chains. There are two N-glycosylation sites in human IgA1-Fc and one in the J-chain. There is no consensus as yet on the functional role of the N-glycosylation. MethodsTo gain a better understanding of their role, we designed a series of IgA1-Fc mutants, which were expressed in the absence or presence of the J-chain. ResultsIgA1-Fc without the J-chain, was predominantly expressed as a monomer, and in its presence dimers and some polymers appeared. N263 (Fc Cα2), N459 (Fc tailpiece) and N49 (J-chain) were shown to be site-specifically modified with N-glycans by mass spectrometry analysis. Mutant IgA1-Fc N459Q failed to form a proper dimer in the presence of the J-chain, instead higher-order aggregates appeared. Fluorescence experiments suggest that the N459-glycans cover a hydrophobic surface at the Fc tailpiece that prevents other Fc molecules from approaching the dimeric IgA. A thermofluor assay revealed that the N-glycans at N263 (Fc) and N49 (J-chain) both contribute in different ways to the thermal stability of the Fc-J-chain complex. NMR analysis of 13C-labeled Fc suggests that the N459-glycan is relatively flexible while the N263-glycan is more rigid. ConclusionsWe conclude that the N459-glycan of IgA1-Fc is essential for dimer formation and prevention of higher-order aggregates while those at N263 (Fc) and N49 (J-chain) stabilize the Fc-J-chain complex. General significanceSite-specific role for N-glycan in molecular assembly is addressed.

Reactivity of NHI-Stabilized Heavier Tetrylenes towards CO2 and N2 O.

A heteroleptic amino(imino)stannylene (TMS2 N)(It BuN)Sn: (TMS=trimethylsilyl, It Bu=C[(N-t Bu)CH]2 ) as well as two homoleptic NHI-stabilized tetrylenes, (It BuN)2 E: (NHI=N-heterocyclic imine, E=Ge, Sn) are presented. VT-NMR investigations of (It BuN)2 Sn: (2) reveal an equilibrium between the monomeric stannylene at room temperature and the dimeric form at -80 °C as well as in the solid state. Upon reaction of the homoleptic tetrylenes with CO2 , both compounds insert two equivalents of CO2 , however differing bonding modes can be observed. (It BuN)2 Sn: (2) inserts one equivalent of CO2 into each Sn-N bond, giving carbamato groups coordinated κ2 O,O' to the metal center. With (It BuN)2 Ge: (3), the Ge-N bonds stay intact upon activation, being bridged by one molecule of CO2 respectively, forming 4-membered rings. Furthermore, the reactivity of 2 towards N2 O was investigated, resulting in partial oxidation to form stannylene dimer [((It BuN)3 SnO)(It BuN)Sn:]2 (6).

Enhanced thermoelectric performances in graphene nanoribbons via BN dimers doping: Theoretical study

Improving the thermoelectric performance of designed graphene nanoribbons is a key stage in the production of thermoelectric nanodevices with many applications. The chemical doping allows armchair graphene nanoribbons (AGNRs) to exhibit controllable thermoelectric characteristics. Here, we use density functional theory-based tight-binding (DFTB) coupled with the non-equilibrium Green's function (NEGF) to study the electronic and thermoelectric properties of AGNR with boron nitride (BN) dimers at room temperature. Changing the concentrations (from 4.17 % (BN)1-structure to 12.5 % (BN)3-structure) and geometrical pattern (ortho, meta, and para form) of BN dimers in the graphene nanoribbons may have a significant effect on the thermoelectric (TE) properties. Our results show that the TE properties of AGNR depend not only on the amount of BN dimers but also on the arrangement of the BN dimers in the AGNR. The thermoelectric figure of merit (ZT) of nanoribbons at room temperature has improved from less than 0.7 to more than 2. These results could be used as an indicator to design nanodevices that have good TE applications.

Temperature-Dependent Spin-Driven Dimerization Determines the Ultrafast Dynamics of a Copper(II)-Bound Tripyrrindione Radical.

Radicals and other open-shell molecules play a central role in chemical transformations and redox chemistry. While radicals are often highly reactive, stable radical systems are desirable for a range of potential applications, ranging from materials chemistry and catalysis to spintronics and quantum information. Here we investigate the ultrafast properties of a stable radical system with temperature-dependent spin-tunable properties. This radical complex, Cu(II) hexaethyl tripyrrin-1,14-dione, accommodates unpaired electrons localized on both the copper metal center and the tripyrrolic ligand. The unusual combination of two unpaired electrons and high stability in this radical molecule enable switchable temperature-dependent spin coupling. Two-dimensional electronic spectroscopy measurements of Cu(II) hexaethyl tripyrrin-1,14-dione were collected at room temperature and at 77 K. At room temperature, the molecules are present as monomers and have short picosecond lifetimes. At 77 K, the molecules are present in a dimer form mediated by ferromagnetic and antiferromagnetic coupling. This reversible spin-driven dimerization changes the optical properties of the system, generating long-lived excitonic states.

Identification and structural analysis of dimeric chicken complement component 3d and its binding with chicken complement receptor 2

Complement component 3d (C3d), the final cleavage product of complement component C3, interacts with CR2 and thus plays a crucial role in linking the innate and adaptive immune systems. Additionally, human C3d executes various functions in its dimeric form, which is more effective than its monomeric form. In this study, we aimed to explored whether chicken C3d (chC3d) exhibits similar characteristics, namely dimerization and binding of dimeric chC3d to chicken CR2 (chCR2). We investigated the interaction and co-localization of chC3d with itself using coimmunoprecipitation and confocal laser scanning microscopy, respectively. Then, dimeric chC3d was detected using native polyacrylamide gel electrophoresis and western blotting, and its equilibrium dissociation constant KD (827 nM) was determined using surface plasmon resonance. Finally, the interaction modes of dimeric chC3d were identified using molecular docking simulations, which revealed that dimeric chC3d could crosslink with chCR2 receptor. Overall, our findings will facilitate future explorations of the chicken complement system.

Oligomerization and tyrosine nitration enhance the allergenic potential of the birch and grass pollen allergens Bet v 1 and Phl p 5.

Protein modifications such as oligomerization and tyrosine nitration alter the immune response to allergens and may contribute to the increasing prevalence of allergic diseases. In this mini-review, we summarize and discuss relevant findings for the major birch and grass pollen allergens Bet v 1 and Phl p 5 modified with tetranitromethane (laboratory studies), peroxynitrite (physiological processes), and ozone and nitrogen dioxide (environmental conditions). We focus on tyrosine nitration and the formation of protein dimers and higher oligomers via dityrosine cross-linking and the immunological effects studied.

Polarization Z-Scan Studies Revealing Plasmon Coupling Enhancement Due to Dimer Formation of Gold Nanoparticles in Nematic Liquid Crystals.

We investigate the plasmon coupling of gold nanoparticle (AuNP) dimers dispersed in a nematic liquid crystal matrix using the polarization z-scan technique. Our experimental setup includes the precise control of incident light polarization through polarization angles of 0°, 45°, and 90°. Two distinct cell orientations are examined: parallel and twisted nematic cells. In parallel-oriented cells, where liquid crystal molecules and AuNPs align with the rubbing direction, we observe a remarkable 2-3-fold increase in the nonlinear absorption coefficient when the polarization of the incident light is parallel to the rubbing direction. Additionally, a linear decrease in the third-order nonlinear absorption coefficient is noted as the polarization angle varies from 0° to 90°. In the case of twisted nematic cells, the NPs do not have any preferred orientation, and the enhancement remains consistent across all polarization angles. These findings conclusively establish that the observed enhancement in the nonlinear absorption coefficient is a direct consequence of plasmon coupling, shedding light on the intricate interplay between plasmonic nanostructures and liquid crystal matrices.

Direct relationship between dimeric form and activity in the acidic copper-zinc superoxide dismutase from lemon.

The copper-zinc superoxide dismutase (CuZnSOD) from lemon (SOD_CL) is active in an acidic environment and resists proteolytic degradation. The enzyme occurs as a dimer, which has an indirect effect on the enzyme activity as the monomer retains only ∼35% of the activity. Here, the crystal structure of SOD_CL at 1.86 Å resolution is reported that may explain this peculiarity. Thecrystal belonged to space group P21, with unit-cell parameters a = 61.11, b=74.55, c = 61.69 Å, β = 106.86°, and contained four molecules in the asymmetric unit. The overall structure of SOD_CL resembles that of CuZnSOD from plants. The structure of SOD_CL shows a unique arrangement of surface loop IV that connects the dimer interface and the active site, which is located away from the dimer-interface region. This arrangement allows direct interaction between the residues residing in the dimer interface and those in the active site. The arrangement also includes Leu62 and Gln164, which are conserved in cytoplasmic CuZnSOD. This supports the classification of SOD_CL as a cytoplasmic CuZnSOD despite sharing the highest amino-acid sequence homology with CuZnSODs from spinach and tomato, which are chloroplastic.

SORLA‐retromer complex formation rely on receptor dimerization within the endosome

AbstractBackgroundSORLA acts as an endosome recycling receptor together with the retromer complex to escort cargo through the endo‐lysosomal tubular network. SORLA is encoded by the SORL1 gene, which is the gene most frequently mutated in patients with Alzheimer’s disease, although it is still not clear which of the many variants are (the most) pathogenic. Here, we studied the biochemistry and cell biology of a SORL1 variant that is found in several European families.MethodsWe used transfected cells to study SORLA maturation and shedding of the soluble sSORLA by Western blotting. Expression of non‐mutated and mutated receptor at the cell surface was determined by flow cytometry, and the intracellular localization and overlap with markers of Endoplasmic reticulum and endosome compartments investigated by immunocytochemistry and confocal microscopy. The cellular distribution of receptor dimerization was visualized using bimolecular fluorescence complementation and proximity ligation assays, and quantified using the GFP‐trap method.ResultsWe showed that non‐mutated SORLA forms a dimer in retromer‐coated endosome structures, while the pathogenic SORLA protein is unable to form dimers, although it traffics to the endosome. This lack of dimerization in the endosome correlates with strongly decreased receptor maturation, expression at the cell surface, and shedding of its luminal sSORLA domain.ConclusionSORLA dimer formation is required for its association with retromer, which is important for efficient receptor maturation and delivery to the cell surface. Decreased cell surface expression of mutated protein with impaired dimerization leads to less shedding, showing how maturation and sSORLA production can reliably inform on the pathogenicity of SORL1 genetic variants in Alzheimer’s disease.