Negatively-charged Residues Research Articles

Uncommon P1 Anchor-featured Viral T Cell Epitope Preference within HLA-A*2601 and HLA-A*0101 Individuals.

The individual HLA-related susceptibility to emerging viral diseases such as COVID-19 underscores the importance of understanding how HLA polymorphism influences peptide presentation and T cell recognition. Similar to HLA-A*0101, which is one of the earliest identified HLA alleles among the human population, HLA-A*2601 possesses a similar characteristic for the binding peptide and acts as a prevalent allomorph in HLA-I. In this study, we found that, compared with HLA-A*0101, HLA-A*2601 individuals exhibit distinctive features for the T cell responses to SARS-CoV-2 and influenza virus after infection and/or vaccination. The heterogeneous T cell responses can be attributed to the distinct preference of HLA-A*2601 and HLA-A*0101 to T cell epitope motifs with negative-charged residues at the P1 and P3 positions, respectively. Furthermore, we determined the crystal structures of the HLA-A*2601 complexed to four peptides derived from SARS-CoV-2 and human papillomavirus, with one structure of HLA-A*0101 for comparison. The shallow pocket C of HLA-A*2601 results in the promiscuous presentation of peptides with "switchable" bulged conformations because of the secondary anchor in the median portion. Notably, the hydrogen bond network formed between the negative-charged P1 anchors and the HLA-A*2601-specific residues lead to a "closed" conformation and solid placement for the P1 secondary anchor accommodation in pocket A. This insight sheds light on the intricate relationship between HLA I allelic allomorphs, peptide binding, and the immune response and provides valuable implications for understanding disease susceptibility and potential vaccine design.

Interaction of Statherin-Derived Peptide with the Surface of Hydroxyapatite: Perspectives Based on Molecular Dynamics Simulations

Introduction: Statherin-derived peptide (StatpSpS) has shown promise against erosive tooth wear. To elucidate its interaction with the hydroxyapatite (HAP) surface, the mechanism related to adsorption of this peptide with HAP was investigated through nanosecond-long all-atom molecular dynamics simulations. Methods: StatpSpS was positioned parallel to the HAP surface in 2 orientations: 1 – neutral and negative residues facing the surface and 2 – positive residues facing the surface. A system containing StatpSpS without HAP was also simulated as control. In the case of systems with HAP, both partially restrained surface and unrestrained surface were constructed. Structural analysis, interaction pattern, and binding-free energy were calculated. Results: In the peptide system without the HAP, there were some conformational changes during the simulation. In the presence of the surface, only moderate changes were observed. Many residues exhibited short and stable distances to the surface, indicating strong interaction. Specially, the residues ASP1 and SER2 have an important role to anchor the peptide to the surface, with positively charged residues, mainly arginine, playing a major role in the further stabilization of the peptide in an extended conformation, with close contacts to the HAP surface. Conclusion: The interaction between StatpSpS and HAP is strong, and the negative charged residues are important to the anchoring of the peptide in the surface, but after the initial placement the peptide rearranges itself to maximize the interactions between positive charged residues.

The exostosin glycosyltransferase 1/STAT3 axis is a driver of breast cancer aggressiveness

The epithelial-mesenchymal transition (EMT) program is crucial for transforming carcinoma cells into a partially mesenchymal state, enhancing their chemoresistance, migration, and metastasis. This shift in cell state is tightly regulated by cellular mechanisms that are not yet fully characterized. One intriguing EMT aspect is the rewiring of the proteoglycan landscape, particularly the induction of heparan sulfate proteoglycan (HSPG) biosynthesis. This proteoglycan functions as a co-receptor that accelerates cancer-associated signaling pathways through its negatively-charged residues. However, the precise mechanisms through which EMT governs HSPG biosynthesis and its role in cancer cell plasticity remain elusive. Here, we identified exostosin glycosyltransferase 1 (EXT1), a central enzyme in HSPG biosynthesis, to be selectively upregulated in aggressive tumor subtypes and cancer cell lines, and to function as a key player in breast cancer aggressiveness. Notably, ectopic expression of EXT1 in epithelial cells is sufficient to induce HSPG levels and the expression of known mesenchymal markers, subsequently enhancing EMT features, including cell migration, invasion, and tumor formation. Additionally, EXT1 loss in MDA-MB-231 cells inhibits their aggressiveness-associated traits such as migration, chemoresistance, tumor formation, and metastasis. Our findings reveal that EXT1, through its role in HSPG biosynthesis, governs signal transducer and activator of transcription 3 (STAT3) signaling, a known regulator of cancer cell aggressiveness. Collectively, we present the EXT1/HSPG/STAT3 axis as a central regulator of cancer cell plasticity that directly links proteoglycan synthesis to oncogenic signaling pathways.

AlphaFold modeling of nepovirus 3C-like proteinases provides new insights into their diverse substrate specificities

The majority of picornaviral 3C proteinases (3Cpro) cleavage sites possess glutamine at the P1 position. Plant nepovirus 3C-like proteinases (3CLpro) show however much broader specificity, cleaving not only after glutamine, but also after several basic and hydrophobic residues. To investigate this difference, we employed AlphaFold to generate structural models of twelve selected 3CLpro, representing six substrate specificities. Generally, we observed favorable correlations between the architecture and charge of nepovirus proteinase S1 subsites and their ability to accept or restrict larger residues. The models identified a conserved aspartate residue close to the P1 residue in the S1 subsites of all nepovirus proteinases examined, consistent with the observed strong bias against negatively-charged residues at the P1 position of nepovirus cleavage sites. Finally, a cramped S4 subsite along with the presence of two unique histidine and serine residues explains the strict requirement of the grapevine fanleaf virus proteinase for serine at the P4 position.

Rational surface charge engineering of haloalkane dehalogenase for boosting the enzymatic performance in organic solvent solutions

Biocatalysis in organic solvents (OSs) has numerous important applications, but native enzymes in OSs often exhibit limited catalytic performance. Herein, we proposed a computation-aided surface charge engineering strategy to improve the catalytic performance of haloalkane dehalogenase DhaA in OSs based on the energetic analysis of substrate binding to the DhaA surface. Several variants with enhanced OS resistance were obtained by replacing negative charged residues on the surface with positive charged residue (Arg). Particularly, a four-substitution variant E16R/E93R/E121R/E257R exhibited the best catalytic performance (five-fold improvement in OS resistance and seven-fold half-life increase in 40% (vol) dimethylsulfoxide). As a result, the overall catalytic performance of the variant could be at least 26 times higher than the wild-type DhaA. Fluorescence spectroscopy and molecular dynamics simulation studies revealed that the residue substitution mainly enhanced OS resistance from four aspects: (a) improved the overall structural stability, (b) increased the hydrophobicity of the local microenvironment around the catalytic triad, (c) enriched the hydrophobic substrate around the enzyme molecule, and (d) lowered the contact frequency between OS molecules and the catalytic triad. Our findings validate that computation-aided surface charge engineering is an effective and ingenious rational strategy for tailoring enzyme performance in OSs.

Molecular mechanism of pH-dependent modulation of Sup35 phase separation by intrinsically disordered M domain.

In budding yeast, starvation stress can trigger a protective response where most cellular processes are downregulated. The transition to this fully reversible state is accompanied by cytoplasmic acidification and subsequent reorganization. Sup35, a translation termination factor with two intrinsically disordered regions (N and M domains), is known to phase separate upon cytoplasmic acidification. Previous work showed the pH dependence of Sup35's phase separation could be attributed to a particularly dense ionizable cluster of negatively-charged residues in the intrinsically disordered M domain, which is proposed to regulate the phase separation properties of the N domain. In this work, we use NMR spectroscopy to probe the pH sensitivity of the ionizable residues in the IDR regions and their interactions. Our aim is to determine the molecular mechanism by which the M domain regulates Sup35's phase separation.

Identification of the Acid-Sensitive Site Critical for Chloral Hydrate (CH) Activation of the Proton-Activated Chloride Channel.

The transmembrane protein TMEM206 was recently identified as the molecular basis of the extracellular proton-activated Cl- channel (PAC), which plays an essential role in neuronal death in ischemia-reperfusion. The PAC channel is activated by extracellular acid, but the proton-sensitive mechanism remains unclear, although different acid-sensitive pockets have been suggested based on the cryo-EM structure of the human PAC (hPAC) channel. In the present study, we firstly identified two acidic amino acid residues that removed the pH-dependent activation of the hPAC channel by neutralization all the conservative negative charged residues located in the extracellular domain of the hPAC channel and some positively charged residues at the hotspot combined with two-electrode voltage-clamp (TEVC) recording in the Xenopus oocytes system. Double-mutant cycle analysis and double cysteine mutant of these two residues proved that these two residues cooperatively form a proton-sensitive site. In addition, we found that chloral hydrate activates the hPAC channel depending on the normal pH sensitivity of the hPAC channel. Furthermore, the PAC channel knock-out (KO) male mice (C57BL/6J) resist chloral hydrate-induced sedation and hypnosis. Our study provides a molecular basis for understanding the proton-dependent activation mechanism of the hPAC channel and a novel drug target of chloral hydrate.SIGNIFICANCE STATEMENT Proton-activated Cl- channel (PAC) channels are widely distributed in the nervous system and play a vital pathophysiological role in ischemia and endosomal acidification. The main discovery of this paper is that we identified the proton activation mechanism of the human proton-activated chloride channel (hPAC). Intriguingly, we also found that anesthetic chloral hydrate can activate the hPAC channel in a pH-dependent manner. We found that the chloral hydrate activates the hPAC channel and needs the integrity of the pH-sensitive site. In addition, the PAC channel knock-out (KO) mice are resistant to chloral hydrate-induced anesthesia. The study on PAC channels' pH activation mechanism enables us to better understand PAC's biophysical mechanism and provides a novel target of chloral hydrate.

Molecular basis of antibiotic self-resistance in a bee larvae pathogen

Paenibacillus larvae, the causative agent of the devastating honey-bee disease American Foulbrood, produces the cationic polyketide-peptide hybrid paenilamicin that displays antibacterial and antifungal activity. Its biosynthetic gene cluster contains a gene coding for the N-acetyltransferase PamZ. We show that PamZ acts as self-resistance factor in Paenibacillus larvae by deactivation of paenilamicin. Using tandem mass spectrometry, nuclear magnetic resonance spectroscopy and synthetic diastereomers, we identified the N-terminal amino group of the agmatinamic acid as the N-acetylation site. These findings highlight the pharmacophore region of paenilamicin, which we very recently identified as a ribosome inhibitor. Here, we further determined the crystal structure of PamZ:acetyl-CoA complex at 1.34 Å resolution. An unusual tandem-domain architecture provides a well-defined substrate-binding groove decorated with negatively-charged residues to specifically attract the cationic paenilamicin. Our results will help to understand the mode of action of paenilamicin and its role in pathogenicity of Paenibacillus larvae to fight American Foulbrood.

Charge substitutions at the voltage-sensing module of domain III enhance actions of site-3 and site-4 toxins on an insect sodium channel

Scorpion α-toxins bind at the pharmacologically-defined site-3 on the sodium channel and inhibit channel inactivation by preventing the outward movement of the voltage sensor in domain IV (IVS4), whereas scorpion β-toxins bind at site-4 on the sodium channel and enhance channel activation by trapping the voltage sensor of domain II (IIS4) in its outward position. However, limited information is available on the role of the voltage-sensing modules (VSM, comprising S1–S4) of domains I and III in toxin actions. We have previously shown that charge reversing substitutions of the innermost positively-charged residues in IIIS4 (R4E, R5E) increase the activity of an insect-selective site-4 scorpion toxin, Lqh-dprIT3-c, on BgNav1-1a, a cockroach sodium channel. Here we show that substitutions R4E and R5E in IIIS4 also increase the activity of two site-3 toxins, LqhαIT from Leiurusquinquestriatus hebraeus and insect-selective Av3 from Anemonia viridis. Furthermore, charge reversal of either of two conserved negatively-charged residues, D1K and E2K, in IIIS2 also increase the action of the site-3 and site-4 toxins. Homology modeling suggests that S2-D1 and S2-E2 interact with S4-R4 and S4-R5 in the VSM of domain III (III-VSM), respectively, in the activated state of the channel. However, charge swapping between S2-D1 and S4-R4 had no compensatory effects on gating or toxin actions, suggesting that charged residue interactions are complex. Collectively, our results highlight the involvement of III-VSM in the actions of both site 3 and site 4 toxins, suggesting that charge reversing substitutions in III-VSM allosterically facilitate IIS4 or IVS4 voltage sensor trapping by these toxins.

Scorpion Toxin-potassium Channel Interaction Law and its Applications

: The scorpion toxins are the largest potassium channel-blocking, peptide family. The understanding of toxin binding interfaces is usually restricted to two classical binding interfaces: one is the toxin α-helix motif, and the other is the antiparallel β-sheet motif. In this review, such traditional knowledge has been updated by another two different binding interfaces: one is BmKTX toxin using the turn motif between the α-helix and antiparallel β-sheet domains as the binding interface, while the other is Tsκ toxin using the turn motif between the β-sheet in the N-terminal and α- helix domains as the binding interface. Their interaction analysis indicated that the scarce, negatively charged residues in the scorpion toxins played a critical role in orientating the toxin binding interface. In view of the toxin, being negatively charged amino acids as a “binding interface regulator”, the law of scorpion toxin-potassium channel interaction was proposed, that is, the polymorphism of negatively charged residue distribution determines the diversity of toxin binding interfaces. Such a law was used to develop the scorpion toxin-potassium channel recognition control technique. According to this technique, three Kv1.3 channel-targeted peptides, using BmKTX as the template, were designed with the distinct binding interfaces from that of BmKTX by modulating the distribution of toxin, negatively charged residues. In view of the potassium channel as the common target of different animal toxins, the proposed law was also shown to adjust the binding interfaces of other animal toxins. The toxin-potassium channel interaction law would strongly accelerate the research and development of different potassium channel-blocking animal toxins in the future.

Quantum Calculations on the Role of Two Amino Acid Groupings that Include Three Positive, Two Negative Charged Residues on the Transport of Protons in the Voltage Sensing Domain of the KV1.2 Potassium Channel

The upper part of the voltage sensing domain (VSD) of the Kv1.2 channel is very similar to the upper part of the Hv1 proton channel, so the transport of protons in the VSD is possible. Our calculations have shown how two sets of five charged amino acids, each with two acid and three basic residues, hence with net one positive charge each, plus three polar but uncharged residues, are critical elements of the proton pathway, together with a limited number of water molecules. The upper group consists of (using numbering from the 3Lut pdb structure) R297,T184,E183,R300,Y266, R303,E226,S176; the lower section includes D259,K306,R309, R240,E236, W232,H310,S169. F233 is an additional critical residue: but rotating it facilitates the motion of a proton through what would otherwise seem a hydrophobic block. An electric field is needed to start the proton cascade from the upper section, which drives protons through the lower section, then along the cytoplasmic surface of the membrane, to the gate. A mutation, F233W, left shifts the gating current curve (MacKinnon and coworkers). In addition to finding the path through the VSD, our calculations explain this left shift, as well as the right shift found with F233E. The calculations show minimal shifts in structure with the field, of the order of 1 Å, but these are enough to produce electrostatic effects that lower the barrier to the proton's progress. Calculations with the protons in several positions have shown the relative energy, and the barriers, to proton transitions along the path. In particular the F233W mutant lowers the activation energy of proton transfer from K306 to D259.

DNA backbone interactions impact the sequence specificity of DNA sulfur-binding domains: revelations from structural analyses

The sulfur atom of phosphorothioated DNA (PT-DNA) is coordinated by a surface cavity in the conserved sulfur-binding domain (SBD) of type IV restriction enzymes. However, some SBDs cannot recognize the sulfur atom in some sequence contexts. To illustrate the structural determinants for sequence specificity, we resolved the structure of SBDSpr, from endonuclease SprMcrA, in complex with DNA of GPSGCC, GPSATC and GPSAAC contexts. Structural and computational analyses explained why it binds the above PT-DNAs with an affinity in a decreasing order. The structural analysis of SBDSpr–GPSGCC and SBDSco–GPSGCC, the latter only recognizes DNA of GPSGCC, revealed that a positively charged loop above the sulfur-coordination cavity electrostatically interacts with the neighboring DNA phosphate linkage. The structural analysis indicated that the DNA–protein hydrogen bonding pattern and weak non-bonded interaction played important roles in sequence specificity of SBD protein. Exchanges of the positively-charged amino acid residues with the negatively-charged residues in the loop would enable SBDSco to extend recognization for more PT-DNA sequences, implying that type IV endonucleases can be engineered to recognize PT-DNA in novel target sequences.

Identification of Sodium Sensitive Site and Chloride Sensitive Site on the C-Terminus of Rat KCNT1 Channel

KCNT1 channel is a sodium and chloride activated potassium channel that is widely expressed in nervous system. Our previous study had identified D818 mutants can dramatically decrease the sodium sensitivity of KCNT1 channel. But the full co-ordinate sodium sensitive site and chloride site of this channel remain unknown. In this abstract, we reported that identification of a sodium binding site of this channel by fully screening 109 negatively charged amino acids mutants on the C-terminus of Slack channel. Results show several mutants of these negative charged channel increased Kd of sodium sensitivity to more than 1 M while several mutants did not show currents. Subsequently, we screened mutants that did not show currents on the background of mutant M335A because the mutant M335A of KCNT1 channel can be activated without intracellular sodium. Thus, we got either macroscopic current or single channel current from all mutants. Combined with electrophysiology experimental data and molecular dynamic simulation, we found that one subunit of Slack channel could bind two sodium ions. One dominant sodium sensitive site is located on RCK1 domain of the Slack channel. Another weak sodium binding pocket that is composed of 6 spatially closed negative charged residues. In the meantime, by molecular dynamic prediction and alignment with C.elegan Slo2 channel, we screened mutants of some positive charged residues on the C-terminus of the rat KCNT1 channel. One chloride binding site was identified from RCK1 domain of KCNT1 channel. In conclusion, our results dissected sodium and chloride sensitive sites of KCNT1 channel.

Immunoinformatics Design of Novel Multi-Epitope Subunit Vaccine for SARS-CoV-2 by Exploring Virus Conserved Sequences of the Spike Glycoproteins

Sars-CoV-2 infection also called COVID-19 is characterized by fever and signs of acute respiratory distress syndrome (ARDS). It is currently a global pandemic with high mortality rate in those with severe disease. Lack of effective vaccine and approved drug for treatment created a disastrous condition among the global communities. This study was designed as a step ahead in the path of protein-based subunit vaccine development. The primary amino acid sequence of SARS-CoV-2 spike glycoprotein was used to design a protein subunit vaccine construct. The molecular weight of vaccine protein was 58.4 kDa with a total number of 8170 atoms and 584 amino acid residues. The theoretical pI was found to be 8.54 showing its slightly basic nature while the total number of negative and positive charged residues were 29 and 33 respectively. The peptide vaccine construct has 147 (25.17%) polar residues and 375 (64.21%) hydrophobic residues. The vaccine construct has cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL) and B cell epitopes of varying lengths having great potential to stimulate high levels of IFN-γ production. It has potent antigenic properties but lacked allergenicity. It is stable and have a good binding affinity for the TLR-4 receptor. In general, this modelling applied a series of immunoinformatics tools in a sequential manner to find an effective vaccine that may be used effectively in fighting against the COVID-19 pandemic. This modelling, however, needs real life experimental validation to prove the workability of the computational work.

Understanding anomalous mobility of proteins on SDS-PAGE with special reference to the highly acidic extracellular domains of human E- and N-cadherins.

During SDS-PAGE experiments, proteins generally display electrophoretic mobility in keeping with their molecular weights; however, some proteins display anomalies in mobility. Here, we focus attention on the anomalies displayed by the highly acidic ∼110 residues-long, sequence-homologous, structurally-analogous, extracellular domains of human E- and N-cadherin. We report that there is a strong correlation between the acidity of each domain and the degree of the anomaly that it displays. The anomaly is only seen if the ratio of the numbers of negatively-charged and positively-charged residues is equal to or greater than the value of 1.50. The degree of the anomaly rises in proportion with this NC:PC ratio. Greater-than-expected anomalies are observed for domains containing dense clusters of negatively charged residues. A simple explanation for these observations is that highly acidic domains electrostatically repel SDS. This results in insufficient SDS binding, insufficient electromotive incentive and (consequently) lowered electrophoretic mobility. This explanation is in consonance with the current view that initial stages of SDS-protein engagement tend to be dominated by electrostatics. We discuss the current anomalies within the broader context of all conceivable explanations for such anomalies.

Structure and function prediction of arsenate reductase from Deinococcus indicus DR1.

Arsenic prevalence in the environment impelled many organisms to develop resistance over the course of evolution. Tolerance to arsenic, either as the pentavalent [As(V)] form or the trivalent form [As(III)], by bacteria has been well studied in prokaryotes, and the mechanism of action is well defined. However, in the rod-shaped arsenic tolerant Deinococcus indicus DR1, the key enzyme, arsenate reductase (ArsC) has not been well studied. ArsC of D. indicus belongs to the Grx-linked prokaryotic arsenate reductase family. While it shares homology with the well-studied ArsC of Escherichia coli having a catalytic cysteine (Cys 12) and arginine triad (Arg 60, 94, and 107), the active site of D.indicus ArsC contains four residues Glu 9, Asp 53, Arg 86, and Glu 100, and with complete absence of structurally equivalent residue for crucial Cys 12. Here, we report that the mechanism of action of ArsC of D. indicus is different as a result of convergent evolution and most likely able to detoxify As(V) using a mix of positively- and negatively-charged residues in its active site, unlike the residues of E. coli. This suggests toward the possibility of an alternative mechanism of As (V) degradation in bacteria.

Substitutions of a buried glutamate residue hinder the conformational change in horse liver alcohol dehydrogenase and yield a surprising complex with endogenous 3′-Dephosphocoenzyme A

Glu-267 is highly conserved in alcohol dehydrogenases and buried as a negatively-charged residue in a loop of the NAD coenzyme binding domain. Glu-267 might have a structural role and contribute to a rate-promoting vibration that facilitates catalysis. Substitutions of Glu-267 with histidine or asparagine residues increase the dissociation constants for the coenzymes (NAD+ by ∼40-fold, NADH by ∼200-fold) and significantly decrease catalytic efficiencies by 16–1200-fold various substrates and substituted enzymes. The turnover numbers modestly change with the substitutions, but hydride transfer is at least partially rate-limiting for turnover for alcohol oxidation. X-ray structures of the E267H and E267 N enzymes are similar to the apoenzyme (open) conformation of the wild-type enzyme, and the substitutions are accommodated by local changes in the structure. Surprisingly, the E267H and E267 N enzymes have endogenous (from the expression in E. coli) 3′-dephosphocoenzyme A bound in the active site with the ADP moiety in the NAD binding site and the pantethiene sulfhydryl bound to the catalytic zinc. The kinetics and crystallography show that the substitutions of Glu-267 hinder the conformational change, which occurs when wild-type enzyme binds coenzymes, and affect productive binding of substrates.

Different Phosphorylation Sites Regulate α1D‐Adrenergic Receptor Function

Human α1D‐adrenergic receptors (α1D‐AR) are seven transmembrane‐spanning proteins that mediate the actions of noradrenaline in homeostasis maintenance as well as the physiology of hypertension and benign prostatic hyperplasia. Although several studies have demonstrated that α1D‐AR is a phosphoprotein, the phosphorylation sites and the specific roles that they play in the function and regulation of this receptor, are currently unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified for the first time distinct phosphorylation patterns on α1D‐AR during noradrenaline (homologous) and non‐related agents‐mediated (heterologous) desensitization. We were able to identify that G protein‐coupled receptor kinase‐2 (GRK2) and protein kinase C α/β (PKC α/β) phosphorylate α1D‐AR during these processes. Phosphorylation of the receptor's intracellular domains, such the intracellular loop 3 or the carboxyl tail, was enough to altered calcium desensitization signals, mitogen activated protein kinases (MAPK) pathway and regulate receptor internalization, suggesting that modular regulatory mechanisms heavily rely on the phosphorylation bar code and the receptor's intracellular domains.Subsequently, we generated six specific non‐phosphorylatable α1D‐AR mutants to delve into the functions of the phosphorylation sites located only at the receptor's carboxyl tail. We found clear differences in the phosphorylation pattern of each mutant during noradrenaline and non‐related agents‐stimulation. Moreover, we analyzed the activation of the MAPK and AKT pathways as well as intracellular calcium mobilization. Modification of any of these phosphorylation sites altered the response when compared to the wild type, but no clear differences were observed among the different α1D‐AR mutants. Interestingly, seven specific phosphorylation sites clustered into two groups in the carboxyl tail, regulate the α1D‐AR subcellular location. This was confirmed when these two clusters were mutated with negative charged residues and the α1D‐AR was located again at the plasma membrane.These data suggest that different phosphorylation sites located in the carboxyl tail regulate the α1D‐AR functions and regulation at different levels. This knowledge highly contribute to unveil new strategies to improve current pharmacological treatments and to avoid drug side effects during sustained administration.Support or Funding InformationPartially supported by Grants from CONACYT (253156 and 882) and DGAPA (IN200915).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Classical Molecular Dynamics Simulation to Understand Role of a Zinc Ion for Aggregation of Amyloid-β Peptides

Metal ions such as those of copper and zinc are considered to accelerate initial formation of amyloid fibril of amyloid-β (Aβ) peptides. In this study, the role of a zinc ion for Aβ peptide aggregation was investigated by the classical molecular dynamics (MD) simulations. The MD results indicated that the negatively-charged residues gained large stabilization in the existence of a zinc ion. On the other hand, histidine and tyrosine which were reported as making a bond with a metal ion were slightly stabled. Therefore, a zinc ion is thought of as combining with histidine or tyrosine after being attracted by negatively-charged residues, because these residues exist near negatively-charged residues. These results indicate that the metal-containing system needs to be treated by quantum-mechanical techniques.

The effect of carboxylate groups on the complexation of metal ion with oligopeptides – Potentiometric investigation

Iron(II), iron(III), cobalt(II), zinc(II) cadmium(II) and lead(II) complexes of Asp2 and Asp3 and other Asp/Glu containing ligands were studied by potentiometry. Our goal was to study the influence of β- and γ-carboxylate groups as well as the increased negative charge of the ligands on the complex formation processes. Mainly 1:1 species are present, the formation of bis(ligand) complexes is not typical for these systems. It can be concluded that the effect of side chain carboxylate groups on the stability of complexes is especially significant in the case of lead(II) and cadmium(II). The greater the number of carboxylic groups there are in the system, the more stable complexes are formed with the studied metal ions. The influence of β-carboxylate groups of aspartic acid is unambiguous, they are directly bounded to the metal ion, which results in increased stability of the complexes. On the other hand, the repulsion between the negative charged residues becomes conspicuous resulting less stable species in the case of peptides containing glutamic acid.