scholarly journals Removing the Polyanionic Cargo Requirement for Assembly of Alphavirus Core-Like Particles to Make an Empty Alphavirus Core

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 846
Author(s):  
Julie M. Button ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Nucleic Acid ◽  
Capsid Protein ◽  
Electrostatic Interactions ◽  
Wild Type ◽  
N Terminus ◽  
Charged Residues ◽  
Size And Morphology ◽  
Positively Charged

The assembly of alphavirus nucleocapsid cores requires electrostatic interactions between the positively charged N-terminus of the capsid protein (CP) and the encapsidated polyanionic cargo. This system differs from many other viruses that can self-assemble particles in the absence of cargo, or form “empty” particles. We hypothesized that the introduction of a mutant, anionic CP could replace the need for charged cargo during assembly. In this work, we produced a CP mutant, Minus 38 (M38), where all N-terminal charged residues are negatively-charged. When wild-type (WT) and M38 CPs were mixed, they assembled into core-like particles (CLPs). These “empty” particles were of similar size and morphology to WT CLPs assembled with DNA cargo, but did not contain nucleic acid. When DNA cargo was added to the assembly mixture, the amount of M38 CP that was assembled into CLPs decreased, but was not fully excluded from the CLPs, suggesting that M38 competes with DNA to interact with WT CPs. The composition of CLPs can be tuned by altering the order of addition of M38 CP, WT CP, and DNA cargo. The ability to produce alphavirus CLPs that contain a range of amounts of encapsidated cargo, including none, introduces a new platform for packaging cargo for delivery or imaging purposes.

Characteristics of the Interaction between Thrombin Exosite1 and the Sequence 269-297 of Platelet Glycoprotein Ibα

1998 ◽  
Vol 80 (08) ◽  
pp. 310-315 ◽  
Author(s):  
Marie-Christine Bouton ◽  
Christophe Thurieau ◽  
Marie-Claude Guillin ◽  
Martine Jandrot-Perrus
Keyword(s):  
Platelet Activation ◽  
Electrostatic Interactions ◽  
Platelet Glycoprotein ◽  
Thrombin Receptor ◽  
Central Position ◽  
Amidolytic Activity ◽  
N Terminus ◽  
Hydrolysis Of ◽  
Chemical Cross Linking ◽  
Positively Charged

SummaryThe interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbα and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbα to interact with thrombin. Three peptides were synthesized, including Ibα 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibα 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibα 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibα 269-287 and α-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when γ-thrombin was substituted for α-thrombin. Ibα 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with α-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibα 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibα 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.

scholarly journals Clusters of Charged Residues at the C Terminus of MotA and N Terminus of MotB Are Important for Function of the Escherichia coli Flagellar Motor

2008 ◽  
Vol 190 (15) ◽  
pp. 5517-5521 ◽  
Author(s):  
Edan R. Hosking ◽  
Michael D. Manson
Keyword(s):  
Escherichia Coli ◽  
Flagellar Motor ◽  
Protein Levels ◽  
Partner Protein ◽  
C Terminus ◽  
N Terminus ◽  
Charged Residues ◽  
Positively Charged ◽  
Terminal Cluster

ABSTRACT MotA contains a conserved C-terminal cluster of negatively charged residues, and MotB contains a conserved N-terminal cluster of positively charged residues. Charge-altering mutations affecting these residues impair motility but do not diminish Mot protein levels. The motility defects are reversed by second-site mutations targeting the same or partner protein.

scholarly journals The C-terminal peptide of CCL21 drastically augments CCL21 activity through the dendritic cell lymph node homing receptor CCR7 by interaction with the receptor N-terminus

2021 ◽  
Author(s):  
Astrid Sissel Jørgensen ◽  
Emma Probst Brandum ◽  
Jeppe Malthe Mikkelsen ◽  
Klaudia A. Orfin ◽  
Ditte Rahbæk Boilesen ◽  
...  
Keyword(s):  
Lymph Node ◽  
Electrostatic Interactions ◽  
Immune Cell ◽  
Direct Interaction ◽  
Receptor Interaction ◽  
Binding Model ◽  
Lymph Node Homing ◽  
Gating Mechanism ◽  
N Terminus ◽  
Positively Charged

AbstractThe endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the main lymph node homing chemokine, but a weak chemo-attractant compared to CCL19. Here we show that the 41-amino acid positively charged peptide, released through C-terminal cleavage of CCL21, C21TP, boosts the immune cell recruiting activity of CCL21 by up to 25-fold and the signaling activity via CCR7 by ~ 100-fold. Such boosting is unprecedented. Despite the presence of multiple basic glycosaminoglycan (GAG) binding motifs, C21TP boosting of CCL21 signaling does not involve interference with GAG mediated cell-surface retention. Instead, boosting is directly dependent on O-glycosylations in the CCR7 N-terminus. As dictated by the two-step binding model, the initial chemokine binding involves interaction of the chemokine fold with the receptor N-terminus, followed by insertion of the chemokine N-terminus deep into the receptor binding pocket. Our data suggest that apart from a role in initial chemokine binding, the receptor N-terminus also partakes in a gating mechanism, which could give rise to a reduced ligand activity, presumably through affecting the ligand positioning. Based on experiments that support a direct interaction of C21TP with the glycosylated CCR7 N-terminus, we propose that electrostatic interactions between the positively charged peptide and sialylated O-glycans in CCR7 N-terminus may create a more accessible version of the receptor and thus guide chemokine docking to generate a more favorable chemokine-receptor interaction, giving rise to the peptide boosting effect.

scholarly journals GFP Fusion to the N-Terminus of MotB Affects the Proton Channel Activity of the Bacterial Flagellar Motor in Salmonella

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1255
Author(s):  
Yusuke V. Morimoto ◽  
Keiichi Namba ◽  
Tohru Minamino
Keyword(s):  
Electrostatic Interactions ◽  
Proton Translocation ◽  
Proton Channel ◽  
Flagellar Motor ◽  
Wild Type ◽  
Over Expression ◽  
N Terminus ◽  
Flow Through ◽  
Proton Leakage ◽  
Bacterial Flagellar Motor

The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-motBfliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-motBfliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA–FliG interaction responsible for torque generation.

Role of the A+ Helix in Heparin Binding to Protein C Inhibitor

1996 ◽  
Vol 75 (05) ◽  
pp. 760-766 ◽  
Author(s):  
Marc G L M Elisen ◽  
Machiel H H Maseland ◽  
Frank C Church ◽  
Bonno N Bouma ◽  
Joost C M Meijers
Keyword(s):  
Protein C ◽  
Electrostatic Interactions ◽  
Deletion Mutant ◽  
Activated Protein C ◽  
Structural Features ◽  
Heparin Binding ◽  
Wild Type ◽  
Protein C Inhibitor ◽  
Positively Charged

SummaryInteractions between proteins and heparin(-like) structures involve electrostatic forces and structural features. Based on charge distributions in the linear sequence of protein C inhibitor (PCI), two positively charged regions of PCI were proposed as possible candidates for this interaction. The first region, the A+ helix, is located at the N-terminus (residues 1-11), whereas the second region, the H helix, is positioned between residues 264 and 280 of PCI. Competition experiments with synthetic peptides based on the sequence of these regions demonstrated that the H helix has the highest affinity for heparin. In contrast to previous observations we found that the A+ helix peptide competed for the interaction of PCI with heparin, but its affinity was much lower than that of the H helix peptide.Recombinant PCI was also used to investigate the role of the A+ helix in heparin binding. Full-length (wild-type) rPCI as well as an A+ helix deletion mutant of PCI (rPCI-Δ2-l 1) were expressed in baby hamster kidney cells and both had normal inhibition activity with activated protein C and thrombin. The interaction of the recombinant PCIs with heparin was investigated and compared to plasma PCI. The A+ helix deletion mutant showed a decreased affinity for heparin in inhibition reactions with activated protein C and thrombin, but had similar association constants compared to wild-type rPCI. The synthetic A+ helix peptide competed with rPCI-Δ2-11 for binding to heparin. This indicated that the interaction between PCI and heparin is fairly non-specific and that the interaction is primarily based on electrostatic interactions.In summary, our data suggest that the H helix of PCI is the main heparin binding region of PCI, but the A+ helix increases the overall affinity for the PCI-heparin interaction by contributing a second positively charged region to the surface of PCI.

scholarly journals Charge-Based Inhibitors of Amylin Fibrillization and Toxicity

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Sharadrao M. Patil ◽  
Andrei T. Alexandrescu
Keyword(s):  
Type 2 Diabetes ◽  
Ionic Strength ◽  
Mouse Model ◽  
Electrostatic Interactions ◽  
Amyloid Fibril ◽  
Structural Models ◽  
Electrostatic Repulsion ◽  
Charged Residues ◽  
Positively Charged

To test the hypothesis that electrostatic repulsion is an important force opposing amyloid fibril assembly, we designed peptides that substitute strings of positively or negatively charged residues into the sequence of the amyloidogenic hormone amylin, which contributes to type 2 diabetes pathology. Arg-1 and Arg-2 substitute four positively charged arginines for segments that in structural models of amylin fibrils form the end of strandβ1 and the beginning of strandβ2, respectively. Mem-T substitutes negatively charged aspartates for the peptide segment with the largest avidity for membranes. All three charge-loaded peptides fibrillize poorly on their own and inhibit fibril elongation of WT-amylin at physiological ionic strength. The inhibition of WT-amylin fibril elongation rates is salt-dependent indicating that the analogs act through electrostatic interactions. Arg-1 protects against WT-amylin cytotoxicity towards a MIN6 mouse model of pancreaticβ-cells, and Arg-2 protects at higher concentrations, whereas Mem-T has no effect. The most effective variant, Arg-1, inhibits WT-amylin fibril elongation rates with an IC50of ~1 µM and cytotoxicity with an IC50of ~50 µM, comparable to other types of fibrillization inhibitors reported in the literature. Taken together, these results suggest that electrostatic interactions can be exploited to develop new types of inhibitors of amyloid fibrillization and toxicity.

Basic Protein Film Methods in the Electron Microscopy of Nucleic Acids

1978 ◽  
Vol 36 (3) ◽  
pp. 587-594
Author(s):  
N. Davidson
Keyword(s):  
Electron Microscopy ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Electrostatic Interactions ◽  
Basic Protein ◽  
Electrolyte Concentration ◽  
Protein Film ◽  
Increased Strength ◽  
Film Method ◽  
Positively Charged

I wish to discuss applications of the basic protein film method for mounting nucleic acids for electron microscopy. In this method, the nucleic acid and an excess of a positively charged low molecular weight globular protein (cytochrome-c is commonly used) are dissolved in a suitable electrolyte solution (ammonium acetate or Tris works well). The electrolyte concentration should be high enough so that the electrostatic binding of the positively charged protein to the negatively charged nucleic acid does not cause precipitation. This solution is layered on to a hypophase which contains a lower concentration of the electrolyte. Some of the protein denatures and forms a film on the surface of the hypophase. Because of the increased strength of the electrostatic interactions at the reduced electrolyte concentration, some of the nucleic acid molecules bind to the positively charged protein film.

scholarly journals Role of the Negative Charges in the Cytosolic Domain of TOM22 in the Import of Precursor Proteins into Mitochondria

1998 ◽  
Vol 18 (6) ◽  
pp. 3173-3181 ◽  
Author(s):  
Frank E. Nargang ◽  
Doron Rapaport ◽  
R. Gary Ritzel ◽  
Walter Neupert ◽  
Roland Lill
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Wild Type ◽  
Binding Studies ◽  
Amino Terminal ◽  
Precursor Proteins ◽  
Mutant Strains ◽  
Cytosolic Domain ◽  
Charged Residues ◽  
Positively Charged

ABSTRACT TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing atom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutanttom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.

scholarly journals Roles of Charged Residues in the C-Terminal Region of PomA, a Stator Component of the Na+-Driven Flagellar Motor

2008 ◽  
Vol 190 (10) ◽  
pp. 3565-3571 ◽  
Author(s):  
Madoka Obara ◽  
Toshiharu Yakushi ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Electrostatic Interactions ◽  
Intramolecular Interactions ◽  
The Other ◽  
Flagellar Motor ◽  
Wild Type ◽  
Mutant Proteins ◽  
E Coli ◽  
Flagellar Motors ◽  
Charged Residues ◽  
Suppressor Analysis

ABSTRACT Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in Escherichia coli. Mutational studies have indicated that the Na+-driven motor of Vibrio alginolyticus may incorporate interactions similar to those of the E. coli motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na+-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na+-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of V. alginolyticus or with the Na+-driven chimeric motor of E. coli. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type V. alginolyticus. The R207E mutant conferred motility with the motor of V. alginolyticus but not with the chimeric motor of E. coli. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in V. alginolyticus but was greatly reduced in E. coli. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex.

scholarly journals Particle Polymorphism Caused by Deletion of a Peptide Molecular Switch in a Quasiequivalent Icosahedral Virus

1998 ◽  
Vol 72 (7) ◽  
pp. 6024-6033 ◽  
Author(s):  
X. Fan Dong ◽  
Padmaja Natarajan ◽  
Mariana Tihova ◽  
John E. Johnson ◽  
Anette Schneemann
Keyword(s):  
Coat Protein ◽  
Single Type ◽  
Flock House Virus ◽  
Wild Type ◽  
Protein Subunits ◽  
Charged Amino Acids ◽  
Irregular Structures ◽  
Icosahedral Virus ◽  
N Terminus ◽  
Positively Charged

ABSTRACT The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.

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