Parallel Dimers Research Articles

Dimerization and lysine substitution of melittin have differing effects on bacteria.

Melittin is a potent antimicrobial peptide from bee venom that is effective against both Gram-positive and Gram-negative bacteria. However, it is extremely toxic to mammalian cells and, as yet, has no clinical use. Modifications to its amino acid sequence, cyclization, truncation, and dimerization have been attempted in order to reduce its toxicity whilst maintaining its antimicrobial activity. In this study, we targeted the three lysine residues present in melittin and substituted them with lysine homologs containing shorter side chains (ornithine, Orn, diaminobutyric acid, Dab, and diaminopropanoic acid, Dap) and made both parallel and antiparallel melittin dimers to observe how lysine substitution and dimerization affects its activity and toxicity. The antibacterial activity of melittin and its analogs was tested against S. aureus (Gram-positive bacteria) and E. coli (Gram-negative bacteria), and cytotoxicity was tested against the mammalian cell lines HEK293 and H4IIE. Overall, dimerization and lysine substitution exhibited improved antimicrobial activity toward E. coli and limited improvement toward S. aureus. However, mammalian cell toxicity was only marginally reduced compared to native melittin. Interestingly, the parallel dimer was found to be marginally more active than the antiparallel dimer, indicating orientation maybe important for activity, although both dimers were less effective than the native and Lys-analog peptides toward S. aureus. Of the Lys substitutions, Dab and Dap improved melittin's activity toward E. coli. Dimerization and Lys substitution of melittin improved the antimicrobial activity toward Gram-negative bacteria but did not significantly improve its activity toward Gram-positive bacteria. Some analogs also displayed reduced toxicity toward HEK293 and H4IIE cells but overall remained toxic at bactericidal concentrations. Our data indicates that although highly antibacterial, melittin's toxicity is the major drawback in its potential use.

Triplet-Triplet Annihilation-Based Photon Upconversion with a Macrocyclic Parallel Dimer.

The integration of multiple chromophore units into a single molecule is expected to improve the performance of photon upconversion based on triplet-triplet annihilation (TTA-UC) that can convert low energy photons to higher energy photons at low excitation intensity. In this study, a macrocyclic parallel dimer of 9,10-diphenylanthracene (DPA) with a precisely parallel orientation, named MPD-2, is synthesized, and its TTA-UC properties are investigated. MPD-2 shows a green-to-blue TTA-UC emission in the presence of a triplet sensitizer, platinum octaethylporphyrin (PtOEP). Compared to monomeric DPA, MPD-2 results in an enhancement of the spin statistical factor of TTA and a decrease in the excitation light intensity due to the intramolecular TTA process. The obtained structure-property relationship provides important information for the further improvement of TTA-UC properties.

Macrocyclic Parallel Dimer Showing Quantum Coherence of Quintet Multiexcitons at Room Temperature.

Singlet fission (SF) is a promising approach in quantum information science because it can generate spin-entangled quintet triplet pairs by photoexcitation independent of temperature. However, it is still challenging to rationally achieve quantum coherence at room temperature, which requires precise control of the orientation and dynamics of triplet pairs. Here we show that the quantum coherence of quintet multiexcitons can be achieved at room temperature by arranging two pentacene chromophores in parallel and in close proximity within a macrocycle. By making dynamic covalent Schiff-base bonds between aldehyde-modified pentacene derivatives, macrocyclic parallel dimer-1 (MPD-1) can be selectively synthesized in a high yield. MPD-1 exhibits fast subpicosecond SF in polystyrene film and generates spin-polarized quintet multiexcitons. Furthermore, the coherence time T2 of the MPD-1 quintet is as long as 648 ns, even at room temperature. This macrocyclic parallel dimer strategy opens up new possibilities for future quantum applications using molecular multilevel qubits.

Multistage protective anti-CelTOS monoclonal antibodies with cross-species sterile protection against malaria

CelTOS is a malaria vaccine antigen that is conserved in Plasmodium and other apicomplexan parasites and plays a role in cell-traversal. The structural basis and mechanisms of CelTOS-induced protective immunity to parasites are unknown. Here, CelTOS-specific monoclonal antibodies (mAbs) 7g7 and 4h12 demonstrated multistage activity, protecting against liver infection and preventing parasite transmission to mosquitoes. Both mAbs demonstrated cross-species activity with sterile protection against in vivo challenge with transgenic parasites containing either P. falciparum or P. vivax CelTOS, and with transmission reducing activity against P. falciparum. The mAbs prevented CelTOS-mediated pore formation providing insight into the protective mechanisms. X-ray crystallography and mutant-library epitope mapping revealed two distinct broadly conserved neutralizing epitopes. 7g7 bound to a parallel dimer of CelTOS, while 4h12 bound to a novel antiparallel dimer architecture. These findings inform the design of antibody therapies and vaccines and raise the prospect of a single intervention to simultaneously combat P. falciparum and P. vivax malaria.

CTMRG study of the critical behavior of an interacting-dimer model

The critical behavior of a dimer model with an interaction favoring parallel dimers in each plaquette of the square lattice is studied numerically using the corner transfer matrix renormalization group algorithm. The critical exponents are known to depend on the chemical potential of vacancies, or monomers. At large average density of the latter, the phase transition becomes the first-order. We compute the scaling dimensions of both the order parameter and temperature in the second-order regime and compare them with the conjecture that the critical behavior is the same as the Ashkin–Teller model on its self-dual critical line.

How irradiation wavelengths affect the regioselectivity in photocycloaddition of 1,4-dihydropyridines

Density functional theory calculations have been performed to reveal the regioselective [2 + 2] and [3 + 2] photocycloaddition of ethyl 3-carboxylate-1,4-diphenyl-1,4-dihydropyridine affected by the irradiation wavelengths. It was found that the regioselectivity is affected by irradiation wavelength, resulting in the formation of the parallel cage dimer diazateraasteranes by [2 + 2] cycloaddition and the orthogonal cage dimer diazatetrakishomocubanes by [3 + 2] cycloaddition. The difference in product formation can be attributed to the distinct mechanism revealed by the DFT/TDDFT analysis at the PCM/M06-2X/def-TZVP//B3LYP/6-31g(d) calculation level. The [2 + 2] photocycloaddition has two steps including an intermolecular and an intramolecular [2 + 2] photocycloaddition, and the [3 + 2] photocycloaddition has three steps including the σ bond formation, the σ bond free rotation and intramolecular reaction. Both the intramolecular reactions of [2 + 2] and [3 + 2] photocycloaddition have the second excitation process of the key intermediates which is related to the irradiation wavelengths and the regioselectivity of the photocycloaddition products.

Directing in Vitro Selection towards G-quadruplex-forming Aptamers to Inhibit HMGB1 Pathological Activity.

In the search for novel, effective inhibitors of High-Mobility Group Box1 (HMGB1)-a protein involved in various inflammatory and autoimmune diseases as well as in cancer-we herein discovered a set of anti-HMGB1 G-quadruplex(G4)-forming aptamers by using an in vitro selection procedure applied to a doped library of guanine-rich oligonucleotides. The selected DNA sequences were then studied in a pseudo-physiological buffer mimicking the extracellular medium, where HMGB1 exerts its pathological activity, using spectroscopic, electrophoretic, and chromatographic techniques. All the oligonucleotides proved to fold into monomeric G4s and in some cases also dimeric species, stable at physiological temperature. Remarkably, the protein preferentially recognized the sequences forming dimeric parallel G4 structures, as evidenced by a properly designed chemiluminescent binding assay which also highlighted a good selectivity of these aptamers for HMGB1. Moreover, all aptamers showed anti-HMGB1 activity, inhibiting protein-induced cell migration. The acquired data allowed identifying L12 as the best anti-HMGB1 aptamer, featured by high thermal and enzymatic stability, no toxicity at least up to 5 μM concentration on healthy cells, along with potent anti-HMGB1 activity (IC50 ca. 28 nM) and good binding affinity for the protein, thus indicating it as a very promising lead candidate for in vivo studies.

Directing in Vitro Selection towards G‐quadruplex‐forming Aptamers to Inhibit HMGB1 Pathological Activity

AbstractIn the search for novel, effective inhibitors of High‐Mobility Group Box1 (HMGB1)—a protein involved in various inflammatory and autoimmune diseases as well as in cancer—we herein discovered a set of anti‐HMGB1 G‐quadruplex(G4)‐forming aptamers by using an in vitro selection procedure applied to a doped library of guanine‐rich oligonucleotides. The selected DNA sequences were then studied in a pseudo‐physiological buffer mimicking the extracellular medium, where HMGB1 exerts its pathological activity, using spectroscopic, electrophoretic, and chromatographic techniques. All the oligonucleotides proved to fold into monomeric G4s and in some cases also dimeric species, stable at physiological temperature. Remarkably, the protein preferentially recognized the sequences forming dimeric parallel G4 structures, as evidenced by a properly designed chemiluminescent binding assay which also highlighted a good selectivity of these aptamers for HMGB1. Moreover, all aptamers showed anti‐HMGB1 activity, inhibiting protein‐induced cell migration. The acquired data allowed identifying L12 as the best anti‐HMGB1 aptamer, featured by high thermal and enzymatic stability, no toxicity at least up to 5 μM concentration on healthy cells, along with potent anti‐HMGB1 activity (IC50 ca. 28 nM) and good binding affinity for the protein, thus indicating it as a very promising lead candidate for in vivo studies.

Multiscale Molecular Dynamics Simulations of the Homodimer Accessory Protein ORF7b of SARS-CoV-2.

The SARS-CoV-2 ORF7b protein has drawn attention for its potential role in viral pathogenesis, but its structural details and lateral membrane associations remain elusive. In this study, we conducted multiscale molecular dynamics simulations to provide detailed molecular insights of the protein's dimerization, which is crucial for unraveling its structural model of protein-protein interface important to regulating cellular immune response. To gain a deeper understanding of homodimer configurations, we employed a machine learning algorithm for structural-based clustering. Clusters were categorized into three distinct groups for both parallel and antiparallel orientations, highlighting the influence of the initial monomer conformation on dimer configurations. Analysis of hydrogen bonding and π-π and π-cation stacking interactions within clusters revealed variations in interactions between clusters. In parallel dimers, weak stacking interactions in the transmembrane (TM) region were observed. In contrast, antiparallel dimers exhibited strong hydrogen bonding and stacking interactions contributing to tight dimeric packing, both within and outside the TM domain. Overall, our study provides a comprehensive view of the structural dynamics of ORF7b homodimerization in both parallel and antiparallel orientations. These findings shed light on the molecular interactions involved in ORF7b dimerization, which are crucial for understanding its potential roles in SARS-CoV-2 pathogenesis. This knowledge could inform future research and therapeutic strategies targeting this viral protein.

A novel interface between the N-terminal and coiled-coil domain of STAT1 functions in an auto-inhibitory manner

BackgroundSTAT1 is an intracellular signaling molecule that is crucially involved in the regulation of the innate immune system by activation of defense mechanisms against microbial pathogens. Phosphorylation-dependent activation of the STAT1 transcription factor is associated with a conversion from an antiparallel to parallel dimer configuration, which after nuclear import binds to DNA. However, not much is known about the specific intermolecular interactions that stabilize unphosphorylated, antiparallel STAT1 complexes prior to activation.ResultsIn this study, we identified a previously unknown interdimeric interaction site, which is involved in the termination of STAT1 signaling. Introduction of the glutamic acid-to-alanine point mutation E169A in the coiled-coil domain (CCD) by site-directed mutagenesis led to increased tyrosine phosphorylation as well as accelerated and prolonged nuclear accumulation in transiently transfected cells. In addition, DNA-binding affinity and transcriptional activity were strongly enhanced in the substitution mutant compared to the wild-type (WT) protein. Furthermore, we have demonstrated that the E169 residue in the CCD mediates the release of the dimer from the DNA in an auto-inhibitory manner.ConclusionBased on these findings, we propose a novel mechanism for the inactivation of the STAT1 signaling pathway, assigning the interface with the glutamic acid residue 169 in the CCD a crucial role in this process.9po3jP83sSjHVE3A4BbhA7Video

Cm-p5 Peptide Dimers Inhibit Biofilms of Candida albicans Clinical Isolates, C. parapsilosis and Fluconazole-Resistant Mutants of C. auris.

Antimicrobial peptides (AMPs) represent a promising class of therapeutic biomolecules that show antimicrobial activity against a broad range of microorganisms, including life-threatening pathogens. In contrast to classic AMPs with membrane-disrupting activities, new peptides with a specific anti-biofilm effect are gaining in importance since biofilms could be the most important way of life, especially for pathogens, as the interaction with host tissues is crucial for the full development of their virulence in the event of infection. Therefore, in a previous study, two synthetic dimeric derivatives (parallel Dimer 1 and antiparallel Dimer 2) of the AMP Cm-p5 showed specific inhibition of the formation of Candida auris biofilms. Here we show that these derivatives are also dose-dependently effective against de novo biofilms that are formed by the widespread pathogenic yeasts C. albicans and C. parapsilosis. Moreover, the activity of the peptides was demonstrated even against two fluconazole-resistant strains of C. auris.

Enhanced Liposomal Drug Delivery Via Membrane Fusion Triggered by Dimeric Coiled-Coil Peptides.

An ideal nanomedicine system improves the therapeutic efficacy of drugs. However, most nanomedicines enter cells via endosomal/lysosomal pathways and only a small fraction of the cargo enters the cytosol inducing therapeutic effects. To circumvent this inefficiency, alternative approaches are desired. Inspired by fusion machinery found in nature, synthetic lipidated peptide pair E4/K4 is used to induce membrane fusion previously. Peptide K4 interacts specifically with E4, and it has a lipid membrane affinity and resulting in membrane remodeling. To design efficient fusogens with multiple interactions, dimeric K4 variants are synthesized to improve fusion with E4-modified liposomes and cells. The secondary structure and self-assembly of dimers are studied; the parallel PK4 dimer forms temperature-dependent higher-order assemblies, while linear K4 dimers form tetramer-like homodimers. The structures and membrane interactions of PK4 are supported by molecular dynamics simulations. Upon addition of E4, PK4 induced the strongest coiled-coil interaction resulting in a higher liposomal delivery compared to linear dimers and monomer. Using a wide spectrum of endocytosis inhibitors, membrane fusion is found to be the main cellular uptake pathway. Doxorubicin delivery results in efficient cellular uptake and concomitant antitumor efficacy. These findings aid the development of efficient delivery systems of drugs into cells using liposome-cell fusion strategies.

Synthesis, Crystal Structure, and Photophysical Properties of Triphenylene 2,3,6,7-Tetracarboxylic Ester-Based Discotic Liquid Crystals

Herein, we report the synthesis and mesomorphic properties of a new series of hexasubstituted triphenylene tetraesters with a mix of four β-alkoxycarbonyl and two adjacent β-alkoxy chains. The key synthetic step involves the efficient fourfold oxidation of methyl groups (CH3) into carboxylic acid (CO2H) functions in the precursor TPCm-(CH3)4 (m = 4, 6, 8, 10) bearing four methyl groups. The crystal structure of the short-chain tetraester TPC4-(COOC4H9)4 showed that two molecules form a parallel slip-stacked dimer with an interplanar spacing of 3.38 Å. Furthermore, two antiparallelly arranged dimers stack up on top of each other to form a one-dimensional columnar assembly with a short interplanar distance of 3.32 Å. Most of the tetrafunctional tetraesters with good solubility exhibit a columnar mesophase that is broad enough between ambient temperature and a moderate clearing temperature of approximately 120 °C, making them attractive for facile processability in future device fabrication. A comparison with structurally related hexaether and hexaester derivatives of triphenylene revealed that these tetrafunctional mesogens had higher clearing temperatures and broader columnar phases. However, similar phase behavior and nearly identical optical properties were observed between the mixed-substituted tetraester and diester. Computational studies at the density functional theory level suggest that increasing the number of peripheral alkoxycarbonyl functions from two to four leads to a reduction in both the HOMO and LUMO levels without affecting the HOMO–LUMO gap.

A study of cyanidin/alginate complexation: Influence of pH in assembly and chiral properties

Cyanidin 3-O-glucoside (CND) is a frequently-used anthocyanin that has excellent antioxidant properties but a limited bioavailability in bloodstream. Complexation of CND with alginate can improve its therapeutic outcome. Here we have studied the complexation of CND with alginate under a range of pH values from 2.5 to 5. CND is positively charged at low pH, and becomes neutral, and then negatively charged as pH increases. CND/alginate complexation was studied by dynamic light scattering, transmission electron microscopy, small angle X-ray scattering, STEM, UV–Vis spectroscopy and circular dichroism (CD). CND/alginate complexes at pH 4.0 and 5.0 form chiral fibres with a fractal structure. At these pH values, CD spectra show very intense bands, which are inverted compared with free CND. Complexation at lower pH results in disordered polymer structures and CD spectra show the same features as for CND in solution. Molecular dynamics simulations suggest the formation of parallel CND dimers through complexation with alginate at pH 3.0, while at pH 4.0 CND dimers form in a cross like arrangement.

Structure of the N-terminal coiled-coil domains of the ciliary protein Rpgrip1l

Rpgrip1l is one of the key ciliary proteins located at the transition zone of the primary cilium, an important organelle for cells to sense the outer environment. Mutations in the RPGRIP1L gene are associated with various ciliopathies. Here, we focused on the N-terminal coiled-coil of Rpgrip1l. By comprehensive biochemical and structural characterizations, we demonstrated that the two predicted coiled-coil regions (CC12) located at Rpgrip1l N-terminus each can form a stable parallel dimer. We further showed that overexpression of Rpgrip1l CC12 in NIH/3T3 cells significantly shortened the length of primary cilia, and this effect depended on the dimer formation. In addition, we found that CC12 of the homolog protein Rpgrip1 in mouse and human were significantly different from Rpgrip1l. Finally, we confirmed that some disease-related mutations can alter the dimeric states of CC12 of Rpgrip1l or Rpgrip1, which might explain the pathogenic mechanisms.

Mutant cancer protein STAT5b has two distinct conformational states.

The point mutation N642H of the signal transducer and activator of transcription 5b (STAT5b) protein is linked to aggressive and drug resistant forms of cancer. While N642H is thought to promote cancer development by inducing constitutive STAT5b activity, the structural and dynamic underpinnings of the mutant's oncogenicity are not well understood. We apply extensive all-atom molecular dynamics simulations to all oligomeric forms of both wild type and N642H STAT5b to elucidate the structural and dynamic differences between the wild type and N642H mutant proteins. Our homology model of the phosphorylated STAT5b parallel dimer reveals a rigid, stable, and compact parallel dimer structure. Our simulations reveal several structural and dynamic similarities between the apo N642H mutant and phosphorylated parallel dimer. In particular, we observe two distinct states in the N642H mutant antiparallel dimer simulations, one of which has similar structure and dynamics to the phosphorylated parallel dimer, indicating that the N642H mutation may bias STAT5b toward a parallel-dimer-like conformation. The structural and dynamic information uncovered in this work contributes to our understanding of STAT5b hyperactivation by N642H and could help pave the way for strategies to overcome the drug resistance of this important mutation.

Metadynamics study of benzyltrimethylammonium binding free energy to EmrE protein in all atom molecular dynamics simulations.

EmrE is a membrane protein found in bacteria such as E. coli that transports positively charged, aromatic toxins outside the cell. It is a parallel dimer and belongs to the small multidrug resistance (SMR) family. Although it is very small, 110 amino acids per monomer, its structure was not resolved at high resolution until recently. The toxin transport is associated with an alternating access movement of the protein monomers. The toxin binds to the protein while the two GLU14 residues of the EmrE are deprotonated in the high pH environment, and is released on the other side when a GLU14 is protonated in low pH. We use metadynamics to calculate the free energy of the binding path of benzyltrimethylammonium to EmrE in a model bacterial membrane. Two geometric reaction coordinates were chosen for the metadynamics runs, distance between center of mass of toxin and the binding site, the angle of the toxin plane with the z-axis. Our results indicate that the free energy of benzyltrimethylammonium binding to EmrE through a water-only path is about −40 kJ/mol. We have also used funnel metadynamics to observe the effect of lipid interaction with the toxin to the binding free energy.

Structure and dynamics of the essential endogenous mycobacterial polyketide synthase PKs13 by cryogenic electron microscopy

Mycobacterium tuberculosis is the leading cause of death by any bacterial infection. We report the structure of the polyketide synthase Pks13 from non-pathogenic Mycobacterium smegmatis, purified from endogenous expression without over expression, and containing its normal substrates ‘trapped in action’. The mycolic acid layer of the cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are therefore front line targets for antimycobacterial drug development. Pks13 is a ∼400-kilodalton module comprised of a parallel dimer of domains, each encoding enzymatic or transport functions that carry out the condensation of two different very long chain fatty acids to produce mycolic acids. Consequently individual enzymatic domains of Pks13 are targets for antimycobacterial drug development. Structures of the multi-domain assembly revealed by cryogenic electron microscopy (cryoEM) define the ketosynthase (KS), linker, and acyltransferase (AT) domains at an overall atomic resolution of 1.8 Å. Focused classification and refinement reveals distinct structures with alternate locations of the N-terminal acyl carrier protein that are ∼60 Å apart, seen at overall resolutions of 2.4-Å and 2.6-Å resolution respectively. These two structures suggest plausible intermediate states on the pathway for substrate delivery from the fatty acyl-ACP ligase (FadD32) to the ketosynthase domain. Other domains visible at lower resolution indicate flexibility relative to the KS-AT core. Domain proximities were probed by chemical cross-linking mass spectrometry. These distances were incorporated into integrative structure modeling to define multiple domain configurations that transport the very long fatty acid chains throughout the multistep Pks13 mediated synthetic pathway. The chemical structures of three bound endogenous long chain fatty acid substrates with their proximal regions defined in the structures were determined by electrospray ionization mass spectrometry.

(2-Hydroxy-3-Methoxybenzylidene)thiazolo[3,2-a]pyrimidines: Synthesis, Self-Assembly in the Crystalline Phase and Cytotoxic Activity.

A series of new 2-hydroxy-3-methoxybenzylidenethiazolo[3,2-a]pyrimidines with different aryl substituents at the 5 position are synthesized and characterized by 1H/ 13C NMR and IR-spectroscopy and mass-spectrometry, as well as single crystal X-ray diffraction (SCXRD). It was demonstrated that the type of hydrogen bonding can play a key role in the chiral discrimination of these compounds in the crystalline phase. The hydrogen bond of the O-H...N type leads to 1D supramolecular heterochiral chains or conglomerate crystallization in the case of the formation of homochiral chains. The hydrogen bond of O-H...O type gave racemic dimers, which are packed into 2D supramolecular layers with a parallel or angular dimers arrangement. Halogen bonding of the N...Br or O...Br type brings a new motif into supramolecular self-assembly in the crystalline phase: the formation of 1D supramolecular homochiral chains instead 2D supramolecular layers. The study of cytotoxicity against various tumor cells in vitro was carried out. It was found that 2-hydroxy-3-methoxybenzylidenethiazolo[3,2-a]pyrimidines with 3-nitrophenyl substituent at C5 carbon atom demonstrated a high efficiency against M-HeLa (cervical adenocarcinoma) and low cytotoxicity against normal liver cells.

Two molecule force spectroscopy on ligand-receptor interactions.

Many biological processes involve the rupture of multiple ligand-receptors or multivalent ligand-receptors. It is challenging to study the rupture of such parallelly arranged multiple ligand-receptors due to the difficulties in engineering such systems in a well-controlled fashion. Here we report the use of two-molecule force spectroscopy to investigate the rupture of two parallelly arranged monomeric streptavidin (mSA)-biotin complexes. By using SpyCatcher-SpyTag chemistry, we successfully engineered a molecular twin of biotin, in which two biotins are arranged in parallel. By reacting mSA with twin biotin, we constructed parallelly arranged two mSA-biotin complexes for force spectroscopy experiments. The incorporation of single molecule fingerprint domains into our mSA-biotin dimers allowed us to identify and assign the rupture events of the parallelly arranged mSA-biotin complexes without any ambiguity in the two-molecule force spectroscopy experiments. Our results revealed that the rupture force of the parallel dimer mSA-biotin is 172 pN at a pulling speed of 400 nm s-1, which is about 1.6 times of that of single mSA-biotin (105 pN). Furthermore, our findings indicate that the two mSA-biotin behave as non-interacting, independent ligand-receptors. The strategy we demonstrated here can be extended to other ligand-receptors and may open up an avenue toward rigorously testing the theoretic predictions proposed in various models regarding the rupture of multiple parallel ligand-receptors.