Glu Residues Research Articles

Molecular docking studies of 1,3,4 -thiadiazoles as myeloperoxidase inhibitors

Myeloperoxidase (MPO) is a heterodimeric, cationic and glycosylated haeme enzyme which gets released under increased oxidative stress producing neutrophil oxidant, hypochlorous acid having the capacity to modify various biomolecules by chlorination and/or oxidation of sulfhydryl groups in proteins causing their inactivation and promoting inflammatory tissue damage. Different levels of hypochlorus acid are used as a trait marker for prescribing the disorders e.g. atherosclerosis, rheumatoid arthritis, lung cancer, Immuno-reactivity. Mini library of 22500 2,5disubstituted 1,3,4 thiadiazoles were docked with Myeloperoxidase in order to identify the potent inhibitor against the enzyme. The chemical nature of the protein and ligands greatly influence the performance of docking process. Keeping this fact in view, critical evaluation of the performance was performed by GLIDE by HTVS, SP and XP. The ADME parameters by QIKPROP and protein-ligand binding free energies were calculated using the Prime/MM-GBSA module of Schrödinger.Both hydrogen bonding and hydrophobic interactions contributed significantly for its ligand binding and core influence the target site through prominent hydrophobic and charged interaction with the backbone and side chain residues in the target site that improves the affinity of the molecule. The compound selected as potent inhibitor is having minimum binding affinity, maximum GScore and minimum FlexX energy. The amino acids residues ASP98, ASP94, THR100 and GLU 102 in the MPO gene domain active site form hydrogen bonds with the ligand. Compounds 3350-5150 showed better interaction with haeme enzyme for further understanding of structures, reliability and Biomolecularactivityy in connection with oxidative stress induced disorders.

Phenothiazines as dual inhibitors of SARS-CoV-2 main protease and COVID-19 inflammation

COVID-19, caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2), currently has no treatment for acute infection. The main protease (Mpro) of SARS-CoV-2 is an essential enzyme for viral replication and an attractive target for disease intervention. The phenothiazine moiety has demonstrated drug versatility for biological systems, including inhibition of butyrylcholinesterase, a property important in the cholinesterase anti-inflammatory cascade. Nineteen phenothiazine drugs were investigated using in silico modelling techniques to predict binding energies and inhibition constants (Ki values) with SARS-CoV-2 Mpro. Because most side-effects of phenothiazines are due to interactions with various neurotransmitter receptors and transporters, phenothiazines with few such interactions were also investigated. All compounds were found to bind to the active site of SARS-CoV-2 Mpro and showed Ki values ranging from 1.30 to 52.4 µM in a rigid active site. Nine phenothiazines showed inhibition constants <10 µM. The compounds with limited interactions with neurotransmitter receptors and transporters showed micromolar (µM) Ki values. Docking results were compared with remdesivir and showed similar interactions with key residues Glu-166 and Gln-189 in the active site. This work has identified several phenothiazines with limited neurotransmitter receptor and transporter interactions and that may provide the dual action of inhibiting SARS-CoV-2 Mpro to prevent viral replication and promote the release of anti-inflammatory cytokines to curb viral-induced inflammation. These compounds are promising candidates for further investigation against SARS-CoV-2.

The Lysozyme Inhibitor Thionine Acetate Is Also an Inhibitor of the Soluble Lytic Transglycosylase Slt35 from Escherichia coli.

Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli.

Computational study on the molecular interactions of tramadol with selected antioxidant and detoxification enzymes in Drosophila melanogaster

Tramadol is a potent analgesic medication prescribed worldwide for treatment of acute and chronic pains. Its relative tendency to be abused has become a public health concern. This study was designed to evaluate the molecular interactions of tramadol with selected antioxidant and detoxification enzymes of Drosophila melanogaster. The structures of CYP2D6, catalase and defense repressor 1 were retrieved from protein database (PubMed, Swiss model) while tramadol 2D structure was obtained from PubChem repository and prepared using LigPrep scripts as implemented in Small-Molecule Drug Discovery Suite of Schrödinger. 2D structure of tramadol was docked into the protein model binding site using Glide software from Schrodinger. The result revealed that, on the binding pocket of CYP2D6, tramadol and CYP2D6 inhibitor bind the protein pocket via hydrogen bond. The hydroxyl group of tramadol and the inhibitor interacts with the C=O groups of the residues Glu 128 and Lys 264 on the protein pocket respectively. Tramadol binds with higher affinity with a docking score of -4.581 kcal/mol when compared with the inhibitor which gave a docking score of -1.865 kcal/mol. With catalase, tramadol and the crystalized ligands were observed to exhibit hydrogen bonding with Ala 64 residue on the protein binding pocket with docking score of -2.348 kcal/mol and -3.431 kcal/mol respectively. The ability of tramadol to interact with these enzymes, strongly suggests that tramadol treatment may induce oxidative stress and at high dose might result in cellular toxicity. Therefore, the toxic effects of tramadol should be of concern despite the important role it plays in pain management.

Cleroda-4(18),13-dien-15,16-olide as novel xanthine oxidase inhibitors: An integrated in silico and in vitro study.

In the present study, in silico predictions and molecular docking were performed on five clerodane diterpenes (1–5) from Polyalthia longifolia seeds to evaluate their potential as xanthine oxidase (XO) inhibitors. The initial screening was conducted by target prediction using TargetNet web server application and only compounds 3 and 4 showed a potential interaction with XO. Compounds 3 and 4 were subsequently subjected to in silico analyses on XO protein structure (PDB: 1N5X) using Schrödinger Release 2020–3 followed by structural modeling & molecular simulation studies to confirm the initial prediction result and identify the binding mode of these compounds to the XO. Molecular docking results revealed that compounds 3 (-37.3 kcal/mol) and 4 (-32.0 kcal/mol) binds more stably to XO than the reference drug allopurinol (-27.0 kcal/mol). Interestingly, two residues Glu 802 and Thr 1010 were observed as the two main H-bond binding sites for both tested compounds and the allopurinol. The center scaffold of allopurinol was positioned by some π-π stacking with Phe 914 and Phe 1009, while that of compounds 3 and 4 were supported by many hydrophobic interactions mainly with Leu 648, Phe 649, Phe 1013, and Leu 1014. Additionally, the docking simulation predicted that the inhibitory effect of compounds 3 and 4 was mediated by creating H-bond with particularly Glu 802, which is a key amino acid for XO enzyme inhibition. Altogether, in vitro studies showed that compounds 3 and 4 had better inhibitory capacity against XO enzyme with IC50 values significantly (p < 0.001) lower than that of allopurinol. In short, the present study identified cleroda-4(18),13-dien-15,16-olide as novel potential XO inhibitors, which can be potentially used for the treatment of gout.

Role of Position K+4 in the Phosphorylation and Dephosphorylation Reaction Kinetics of the CheY Response Regulator.

Two-component signaling is a primary method by which microorganisms interact with their environments. A kinase detects stimuli and modulates autophosphorylation activity. The signal propagates by phosphotransfer from the kinase to a response regulator, eliciting a response. Response regulators operate over a range of time scales, corresponding to their related biological processes. Response regulator active site chemistry is highly conserved, but certain variable residues can influence phosphorylation kinetics. An Ala-to-Pro substitution (K+4, residue 113) in the Escherichia coli response regulator CheY triggers a constitutively active phenotype; however, the A113P substitution is too far from the active site to directly affect phosphochemistry. To better understand the activating mechanism(s) of the substitution, we analyzed receiver domain sequences to characterize the evolutionary role of the K+4 position. Although most featured Pro, Leu, Ile, and Val residues, chemotaxis-related proteins exhibited atypical Ala, Gly, Asp, and Glu residues at K+4. Structural and in silico analyses revealed that CheY A113P adopted a partially active configuration. Biochemical data showed that A113P shifted CheY toward a more activated state, enhancing autophosphorylation. By characterizing CheY variants, we determined that this functionality was transmitted through a hydrophobic network bounded by the β5α5 loop and the α1 helix of CheY. This region also interacts with the phosphodonor CheAP1, suggesting that binding generates an activating perturbation similar to the A113P substitution. Atypical residues like Ala at the K+4 position likely serve two purposes. First, restricting autophosphorylation may minimize background noise generated by intracellular phosphodonors such as acetyl phosphate. Second, optimizing interactions with upstream partners may help prime the receiver domain for phosphorylation.

Roles of constitutive and signal-dependent protein phosphatase 2A docking motifs in burst attenuation of the cyclic AMP response element-binding protein

The cAMP response element-binding protein (CREB) is an important regulator of cell growth, metabolism, and synaptic plasticity. CREB is activated through phosphorylation of an evolutionarily conserved Ser residue (S133) within its intrinsically disordered kinase-inducible domain (KID). Phosphorylation of S133 in response to cAMP, Ca2+, and other stimuli triggers an association of the KID with the KID-interacting (KIX) domain of the CREB-binding protein (CBP), a histone acetyl transferase (HAT) that promotes transcriptional activation. Here we addressed the mechanisms of CREB attenuation following bursts in CREB phosphorylation. We show that phosphorylation of S133 is reversed by protein phosphatase 2A (PP2A), which is recruited to CREB through its B56 regulatory subunits. We found that a B56-binding site located at the carboxyl-terminal boundary of the KID (BS2) mediates high-affinity B56 binding, while a second binding site (BS1) located near the amino terminus of the KID mediates low affinity binding enhanced by phosphorylation of adjacent casein kinase (CK) phosphosites. Mutations that diminished B56 binding to BS2 elevated both basal and stimulus-induced phosphorylation of S133, increased CBP interaction with CREB, and potentiated the expression of CREB-dependent reporter genes. Cells from mice harboring a homozygous CrebE153D mutation that disrupts BS2 exhibited increased S133 phosphorylation stoichiometry and elevated transcriptional bursts to cAMP. These findings provide insights into substrate targeting by PP2A holoenzymes and establish a new mechanism of CREB attenuation that has implications for understanding CREB signaling in cell growth, metabolism, synaptic plasticity, and other physiologic contexts.

Reverse Protonation of Buried Ion-Pairs in Staphylococcal Nuclease Mutants.

Although buried titratable residues in protein cavities are often of major functional importance, it is generally challenging to understand their properties such as the ionization state and factors of stabilization based on experimental studies alone. A specific set of examples involve buried Glu-Lys pairs in a series of variants of Staphylococcal nuclease, for which recent structural and thermodynamic studies appeared to suggest that both the stability and the ionization state of the buried Glu-Lys pair are sensitive to its orientation (i.e., Glu23-Lys36 vs Lys23-Glu36). To further clarify the situation, especially ionization states of the buried Glu-Lys pairs, we have conducted extensive molecular dynamics simulations and free energy computations. Microsecond molecular dynamics simulations show that the hydration level of the cavity depends on the orientation of the buried ion-pair therein as well as its ionization state; free energy simulations recapitulate the relative stability of Glu23-Lys36 (EK) vs Lys23-Glu36 (KE) mutants measured experimentally, although the difference is similar in magnitude regardless of the ionization state of the Glu-Lys pair. A complementary set of free energy simulations strongly suggests that, in contrast to the original suggestion in the experimental analysis, the Glu and Lys residues prefer to adopt their charge-neutral rather than the ionized states. This result is consistent with the low dielectric constant computed for water in the cavity, which makes it difficult for the protein cavity to stabilize a pair of charged Glu-Lys residues, even with water penetration. The current study highlights the role of free energy simulations in understanding the ionization state of buried titratable residues and the relevant energetic contributions, forming the basis for the rational design of buried charge networks in proteins.

The Role of Conserved Residues in the DEDDh Motif: the Proton-Transfer Mechanism of HIV-1 RNase H

RNase H is a prototypical example for two-metal-ion catalysis in enzymes. An RNase H activity cleaving the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid is present not only in important drug targets, such as the HIV-1 reverse transcriptase, but also in many other nucleases, such as Homo sapiens (Hs) and Escherichia coli (Ec) RNase H or, notably, in enzymes that are part of the CRISPR gene editing molecular machinery. Despite its importance, the reaction mechanism uncovering the proton-transfer events is not yet understood. In particular, it is not known, which group is the proton donor for the leaving group. Moreover, several different proton acceptors were proposed, and the exact identity of the proton acceptor is also elusive. Here, we revisit the mechanism for RNAse H, whereby we find that the highly conserved Glu residue of the DDE motif acts as a proton donor via a mechanism further stabilized by the 2′O atom of the sugar. Additionally, we also describe an alternative proton-transfer mechanism via a conserved catalytic His residue to deprotonate the attacking water molecule. Furthermore, our quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method provide an accurate free-energy profile for the reaction catalyzed by the HIV-1 RNase H. Our reported pathway is consistent with kinetic data obtained for mutant HIV-1, Hs, and Ec RNase H, with the calculated pKa values of the DEDD residues and with crystallographic studies. The overall reaction barrier of ∼19 kcal mol–1, encountered in the phosphate-cleavage step, matches the slow experimental rate of ∼1–100 min–1. Additionally, using molecular dynamics (MD) calculations, we sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Taken together, we provide a molecular mechanism of the nuclease catalytic reaction that is likely common for the broad family of two-metal-ion catalytic phosphate-cleaving enzymes with a DDE motif.

Developing and Assessing Nonbonded Dummy Models of Magnesium Ion with Different Hydration Free Energy References.

A large diversity in the targeted hydration free energies (HFEs) during model parameterization of metal ions was reported in the literature with a difference by dozens of kcal/mol. Here, we developed a series of nonbonded dummy models of the Mg2+ ion targeting different HFE references in TIP3P water, followed by assessments of the designed models in the simulations of MgCl2 solution and biological systems. Together with the comparison of existing models, we conclude that the difference in the targeted HFEs has a limited influence on the model performance, while the usability of these models differs from case to case. The feasibility of reproducing more properties of Mg2+ such as diffusion constants and water exchange rates using a nonbonded dummy model is demonstrated. Underestimated activity derivative and osmotic coefficient of MgCl2 solutions in high concentration reveal a necessity for further optimization of ion-pair interactions. The developed dummy models are applicable to metal coordination with Asp, Glu, and His residues in metalloenzymes, and the performance in predicting monodentate or bidentate binding modes of Asp/Glu residues depends on the complexity of metal centers and the choice of protein force fields. When both the binding modes coexist, the nonbonded dummy models outperform point charge models, probably in need of considering polarization of metal-binding residues by, for instance, charge calibration in classical force fields. This work is valuable for the use and further development of magnesium ion models for simulations of metal-containing systems with good accuracy.

Safety enhancement of a genetically modified live rabies vaccine strain by introducing an attenuating Leu residue at position 333 in the glycoprotein

To improve the safety of genetically modified live rabies vaccine strains, most studies have utilized an attenuating Arg-to-Glu mutation at position 333 in the glycoprotein (G333), which is responsible for attenuation of the live vaccine strain SAG2. The Glu residue requires two nucleotide substitutions to revert to pathogenic Arg, thus significantly lowering the probability of pathogenic reversion caused by the Glu-to-Arg mutation at G333. However, only one nucleotide substitution is sufficient to convert the Glu residue to another pathogenic residue, Lys, and thereby to cause pathogenic reversion. This indicates a potential safety problem of SAG2 and the live vaccine candidates attenuated by Glu at G333. In this study, aiming to solve this problem, we examined the utility of a Leu residue, which requires two nucleotide substitutions to be both Arg and Lys, as an attenuating mutation at G333. Using a reverse genetics system of the live vaccine strain ERA, we generated ERA-G333Leu by introducing an Arg-to-Leu mutation at G333. Similar to ERA-G333Glu, which is attenuated by an Arg-to-Glu mutation at G333, ERA-G333Leu did not cause obvious clinical signs in 6-week-old mice after intracerebral inoculation. Importantly, after 10 passages in suckling mouse brains, ERA-G333Glu acquired a pathogenic Lys or Arg at G333 and a high level of lethality in mice, whereas ERA-G333Leu retained the attenuating Leu at G333 and only showed a modest level of virulence probably caused by a mutation at G194. In addition, ERA-G333Leu and ERA-G333Glu induced neutralizing antibody response and protective immunity in mice with similar efficiencies. The results demonstrate that, compared to ERA-G333Glu, ERA-G333Leu is more stably attenuated, also indicating the high utility of a Leu residue as an attenuating mutation at G333 in the development of live rabies vaccine strains with a high level of safety.

Binding interactions of acrylamide with lysozyme and its underlying mechanisms based on multi-spectra, isothermal titration microcalorimetry and docking simulation

Acrylamide is a known carcinogenic and neurotoxic organic compound that is widespread in the environment and poses potential threats to human beings and other living organisms. Herein, we explored the interaction mechanism of acrylamide with lysozyme via multiple spectroscopic approaches, ITC (isothermal titration calorimetry), enzyme activity, as well as molecular docking under physiological conditions. Results suggested that acrylamide can interact with lysozyme fluorophore residues including Trp and Tyr, and exhibit a fluorescence sensitization ability toward these amino acids. The direct interaction of lysozyme with acrylamide resulted in the unfolding of polypeptide chains along with the loosening of protein skeletons, decreasing the α-helical content, and hence a smaller size of lysozyme agglomerated. Moreover, acrylamide docked to lysozyme at relatively low affinity and the interactions were primarily driven by van der Waals and hydrogen bonding interactions. Further molecular analyses illustrated that the binding site was around the active center of lysozyme, acrylamide interacted with Glu 35, Asn 44, Asn 46, Asp 52, Gln 57, Ala 107, Trp 108 and Val 109. The molecular lysozyme activity was repressed due to the direct cross talk of acrylamid with the key residues Glu 35, as well as Asp 52 of the enzyme activity near the active center lysozyme. In addition, the fluorophore residues Trp 108 was also in the binding pocket, which can also explain the fluorescence sensitization phenomenon. Taken together, our study demonstrated the structure and function of lysozyme were both affected by acrylamide.

Analysis of Binding Modes of Antigen-Antibody Complexes by Molecular Mechanics Calculation.

Antibodies are one of the most important protein molecules in biopharmaceutics. Due to the recent advance in technology for producing monoclonal antibodies, many structural data are available on the antigen-antibody complexes. To characterize the molecular interaction in antigen-antibody recognition, we computationally analyzed 500 complex structures by molecular mechanics calculations. The presence of Ser and Tyr is markedly large in the complementarity-determining regions (CDRs). Although Ser is abundant in CDRs, its contribution to the binding score is not large. Instead, Tyr, Asp, Glu, and Arg significantly contribute to the molecular interaction from the viewpoint of the binding score. The decomposition of the binding score suggests that the hydrophilic interaction is predominant in all CDRs compared with the hydrophobic one. The contribution of the heavy chain is larger than that of the light chain. In particular, H2 and H3 largely contribute to the binding interaction. Tyr is a main contributing residue both in H2 and H3. The positively charged residue Arg also significantly contributes to the binding score in H3, while the contribution of Lys is small. The appearance of Ser is remarkable in H2, and Asp is abundant in H3. The non-charged polar residues, Thr, Asn, and Gln, appear much in H2, compared to appearing in H3. The negatively charged residues Asp and Glu significantly contribute to the binding score in H3. The contributions of Phe and Trp are not large in spite that the aromatic residues are capable of making the π-π or CH-π interaction. Gly is commonly abundant both in H2 and H3. The average distance of the shortest direct hydrogen bond between the antigen and antibody is longer than that of the hydrogen bonds observed in the complexes between compounds and their target proteins. Therefore, the antigen-antibody interface is not so tight as the compound-target protein interface. The calculation of shape complementarity is consistent with the result of the hydrogen bonds in that the fitness of the antigen-antibody contact is not so high as that of the compound-target protein contact. There exist many water molecules at the antigen-antibody interface. These findings suggest that Tyr, Asp, Glu, and Arg are rich in H3 and work as major contributors for the interaction with the antigen. Ser, Thr, Asn, and Gln are rich in H2 and support the interaction with enhancing molecular fitness. Gly is helpful in increasing flexibility and geometrical diversity. Because the antigen-antibody binding is fundamentally hydrophilic-driven, the non-polar residues are unfavorable for mediating the contact even for the aromatic residues such as Phe and Trp.

Evolutionary adaptation of the γ‐carboxyglutamic acid domain in blood coagulation factor X in the Serpentes suborder

The serine protease factor Xa plays an important role in blood coagulation as it, in complex with its cofactor Va, proteolytically activates prothrombin to thrombin, the latter being a key enzyme in coagulation. To perform this vital function, factor Xa undergoes numerous posttranslational modifications including vitamin K-dependent γ-carboxylation of glutamic acid (Glu) residues into so-called Gla residues. A correctly γ-carboxylated GLA domain is essential for interaction with the negatively charged membrane surface of activated platelets or endothelial cells at the site of injury where clot formation is required. Moreover, assembly of factor Xa with the cofactor Va into the prothrombinase enzyme complex occurs on a negatively charged membrane surface only. Interestingly, the venom of some Australian Elapid snakes comprises powerful prothrombin-activating enzyme complexes consisting of factor Xa- and Va-like proteins that are specifically expressed in the venom gland. We have previously demonstrated that this venom enzyme complex is capable of functioning independently of a negatively charged phospholipid membrane. We aim to uncover the putative differential role of the GLA domain between human and snake venom (Pseudonaja textilis, pt) factor Xa. To do so, we exchanged the human factor Xa (hFX) GLA domain for that of the snake venom ortholog (ptFX), thereby generating hFX-wt, hFX-ptGLA, ptFX-wt, ptFX-hGLA. The FX variants were recombinantly expressed and purified employing chromatography techniques. FX activation by either the human extrinsic or intrinsic coagulation tenase complex was analyzed using a peptidyl substrate specific for activated FX. Introduction of the ptGLA domain into hFX modestly improved the catalytic rate of the extrinsic reaction, while for the intrinsic reaction the substrate binding affinity was enhanced. Conversely, using similar conditions no FXa formation could be detected following incubation of ptFX or ptFX-hGLA with the human intrinsic or extrinsic tenase complex. Assessment of the FX-specific plasma clotting activity in which thrombin-dependent fibrin clot formation is monitored following either an extrinsic or intrinsic coagulation trigger revealed that while the extrinsic clotting activity of hFX was drastically reduced when hGLA was exchanged for ptGLA (3% residual activity), its intrinsic activity was unperturbed. Use of human versus rabbit reagents for the extrinsic pathway may explain the different observations. Conversely, exchange of ptGLA for hGLA resulted in an overall substantial increase in the FX-specific clotting activity of ptFX-hGLA. Finally, exchanging the ptGLA domain for the hGLA domain did not abolish the requirement for a negatively charged membrane surface in Va-dependent prothrombin activation. These data indicate that the snake venom GLA domain interacts with the intrinsic tenase complex and contributes to substrate conversion and subsequent thrombin formation, which may point to evolutionary adaptation of this snake venom GLA domain.

Molecular Characterization of Complement Component 3 (C3) in the Pearl Oyster Pinctada fucata Improves Our Understanding of the Primitive Complement System in Bivalve.

As the central component in the complement system, complement component 3 (C3) plays essential roles in both the innate and adaptive immune responses. Here, a C3 gene (designated as pf-C3) was obtained from the pearl oyster Pinctada fucata by RT-PCR and rapid amplification of cDNA ends (RACE). The pf-C3 cDNA consists of 5,634 bp with an open reading frame (ORF) of 5,193 bp encoding a protein of 1,730 amino acids with a 19 residue signal peptide. The deduced pf-C3 protein possessed the characteristic structural features present in its homologs and contained the A2M_N_2, ANATO, A2M, A2M_comp, A2M_recep, and C345C domains, as well as the C3 convertase cleavage site, thioester motif, and conserved Cys, His, and Glu residues. Phylogenetic analysis revealed that pf-C3 is closely related to the C3s from other mollusks. Pf-C3 mRNA was expressed in all examined tissues including gill, digestive gland, adductor muscle, mantle and foot, while the highest expression was found in the digestive gland. Following the challenge with Vibrio alginolyticus, pf-C3 expression was significantly induced in hemocytes. Luciferase reporter assays indicated that pf-C3a could activate the NF-κB signal pathway in HEK293T cells. Further knockdown of pf-C3 by specific siRNA could significantly reduce the phagocytosis of V. alginolyticus by hemocytes in vitro. These results would help increase understanding of the function of C3 in the invertebrate immune system and therefore provide new insights into the roles of the primitive complement system in invertebrates.

Attenuation of Human Lysozyme Amyloid Fibrillation by ACE Inhibitor Captopril: A Combined Spectroscopy, Microscopy, Cytotoxicity, and Docking Study.

Misfolding proteins could form oligomers or amyloid fibers, which can cause a variety of amyloid-associated diseases. Thus, the inhibition of protein misfolding and fibrillation is a promising way to prevent and treat these diseases. Captopril (CAP) is an angiotensin-converting enzyme inhibitor (ACEI) that is widely used to treat diseases such as hypertension and heart failure. In this study, we found that CAP inhibits human lysozyme (HL) fibrillation through the combination techniques of biophysics and biochemistry. The data obtained by thioflavin-T (ThT) and Congo red (CR) assays showed that CAP hindered the aggregation of HL amyloid fibrils by reducing the β-sheet structure of HL amyloid, with an IC50 value of 34.75 ± 1.23 μM. Meanwhile, the particle size of HL amyloid decreased sharply in a concentration-dependent approach after CAP treatment. According to the visualization of atomic force microscopy (AFM) and transmission electron microscopy (TEM), we verified that in the presence of CAP, the needle-like fibers of HL amyloid were significantly reduced. In addition, CAP incubation dramatically improved the cell survival rate exposed to HL fibers. Our studies also revealed that CAP could form hydrogen bonds with amino acid residues of Glu 35 and Ala 108 in the binding pocket of HL, which help in maintaining the α-helical structure of HL and then prevent the formation of amyloid fibrillation. It can be concluded that CAP has antiamyloidogenic activity and a protective effect on HL amyloid cytotoxicity.

Inhibition of SARS-CoV-2 main protease: a repurposing study that targets the dimer interface of the protein

Coronavirus disease-2019 (COVID-19) was firstly reported in Wuhan, China, towards the end of 2019, and emerged as a pandemic. The spread and lethality rates of the COVID-19 have ignited studies that focus on the development of therapeutics for either treatment or prophylaxis purposes. In parallel, drug repurposing studies have also come into prominence. Herein, we aimed at having a holistic understanding of conformational and dynamical changes induced by an experimentally characterized inhibitor on main protease (Mpro) which would enable the discovery of novel inhibitors. To this end, we performed molecular dynamics simulations using crystal structures of apo and α-ketoamide 13b-bound Mpro homodimer. Analysis of trajectories pertaining to apo Mpro revealed a new target site, which is located at the homodimer interface, next to the catalytic dyad. Thereafter, we performed ensemble-based virtual screening by exploiting the ZINC and DrugBank databases and identified three candidate molecules, namely eluxadoline, diosmin, and ZINC02948810 that could invoke local and global conformational rearrangements which were also elicited by α-ketoamide 13b on the catalytic dyad of Mpro. Furthermore, ZINC23881687 stably interacted with catalytically important residues Glu166 and Ser1 and the target site throughout the simulation. However, it gave positive binding energy, presumably, due to displaying higher flexibility that might dominate the entropic term, which is not included in the MM-PBSA method. Finally, ZINC20425029, whose mode of action was different, modulated dynamical properties of catalytically important residue, Ala285. As such, this study presents valuable findings that might be used in the development of novel therapeutics against Mpro. Communicated by Ramaswamy H. Sarma

Sequence-Selective Decapeptide Synthesis by the Parallel Operation of Two Artificial Molecular Machines

We report on the preparation of a decapeptide through the parallel operation of two rotaxane-based molecular machines. The synthesis proceeds in four stages: (1) simultaneous operation of two molecular peptide synthesizers in the same reaction vessel; (2) selective residue activation of short-oligomer intermediates; (3) ligation; (4) product release. Key features of the machine design include the following: (a) selective transformation of a thioproline building block to a cysteine (once it has been incorporated into a hexapeptide intermediate by one molecular machine); (b) a macrocycle-peptide hydrazine linkage (as part of the second machine) to differentiate the intermediates and enable their directional ligation; and (c) incorporation of a Glu residue in the assembly module of one machine to enable release of the final product while simultaneously removing part of the assembly machinery from the product. The two molecular machines participate in the synthesis of a product that is beyond the capability of individual small-molecule machines, in a manner reminiscent of the ligation and post-translational modification of proteins in biology.

Lipid-Dependent Titration of Glutamic Acid at a Bilayer Membrane Interface.

The ionization properties of protein side chains in lipid-bilayer membranes will differ from the canonical values of side chains exposed to an aqueous solution. While the propensities of positively charged side chains of His, Lys, and Arg to release a proton in lipid membranes have been rather well characterized, the propensity for a negatively charged Glu side chain to receive a proton and achieve the neutral state in a bilayer membrane has been less well characterized. Indeed, the ionization of the glutamic acid side chain has been predicted to depend on its depth of burial in a lipid membrane but has been difficult to verify experimentally. To address the issue, we incorporated an interfacial Glu residue at position 4 of a distinct 23-residue transmembrane helix and used 2H NMR to examine the helix properties as a function of pH. We observe that the helix tilt and azimuthal rotation vary little with pH, but the extent of helix unraveling near residues 3 and 4 changes as the Glu residue E4 titrates. Remarkably, the 2H quadrupolar splitting for the side chain of alanine A3 responds to pH with an apparent pKa of 4.8 in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 6.3 in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), but is unchanged up to pH 8.0 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of residue E4. With bilayers composed of alkali-stable ether-linked lipids, the side chain of A3 responds to pH with an apparent pKa of 11.0 in the ether analogue of DOPC. These results suggest that the depth dependence of Glu ionization in lipid-bilayer membranes may be steeper than previously predicted or envisioned.

Characterization of 2 linear peptides without disulfide bridges from the venom of the spider Lycosa poonaensis (Lycosidae).

Spider venom is a complex mixture of bioactive components, in which peptides play an important role by showing neurotoxicity or cytotoxicity. Disulfide-rich peptides are major components in the venom, but linear peptides without disulfide bridges are also present and often show antimicrobial activity. In this study, we analyzed the venom of the spider Lycosa poonaensis (Lycosidae) to find novel antimicrobial peptides using mass spectrometry. The result revealed that 120 out of 401 detected components were nondisulfide-bridged peptides. From them, the sequence of 2 peptides (lyp2370 and lyp1987) were determined by MS/MS analysis. The biological activity test revealed that lyp2370 has only weak antibacterial activity. On the other hand, lyp1987, which is identical to M-lycotoxin-Ls3b from the Lycosa singoriensi venom, showed significant antibacterial activity. The weak activity of lyp2370 was found to be due to the presence of a Glu residue on the hydrophilic face of its amphipathic α-helical structure.