scholarly journals The C Terminus of Component C2II ofClostridium botulinum C2 Toxin Is Essential for Receptor Binding

2000 ◽  
Vol 68 (8) ◽  
pp. 4566-4573 ◽  
Author(s):  
Dagmar Blöcker ◽  
Holger Barth ◽  
Elke Maier ◽  
Roland Benz ◽  
Joseph T. Barbieri ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Receptor Binding ◽  
Sodium Dodecyl ◽  
Cytotoxic Action ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Terminal Amino ◽  
Function Relationship ◽  
C2 Toxin

ABSTRACT The binary Clostridium botulinum C2 toxin consists of two separate proteins, the binding component C2II (80.5 kDa) and the actin-ADP-ribosylating enzyme component C2I (49.4 kDa). For its cytotoxic action, C2II binds to a cell membrane receptor and induces cell entry of C2I via receptor-mediated endocytosis. Here we studied the structure-function relationship of C2II by constructing truncated C2II proteins and producing polyclonal antisera against selective regions of C2II. An antibody raised against the C terminus (amino acids 592 to 721) of C2II inhibited binding of C2II to cells. The antibody prevented pore formation by C2II oligomers in artificial membranes but did not influence the properties of existing channels. To further define the region responsible for receptor binding, we constructed proteins with deletions in C2II; specifically, they lacked amino acid residues 592 to 721 and the 7 C-terminal amino acid residues. The truncated proteins still formed sodium dodecyl sulfate-stable oligomers but were unable to bind to cells. Our data indicate that the C terminus of C2II mediates binding of the protein to cells and that the 7 C-terminal amino acids are structurally important for receptor binding.

scholarly journals Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 715
Author(s):  
Tamara Tomanić ◽  
Claire Martin ◽  
Holly Stefen ◽  
Esmeralda Parić ◽  
Peter Gunning ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Growth Cones ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
C Terminus ◽  
Primary Mouse ◽  
Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

scholarly journals SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure

2019 ◽  
Vol 24 (9) ◽  
pp. 928-938 ◽  
Author(s):  
Luca Palazzolo ◽  
Chiara Paravicini ◽  
Tommaso Laurenzi ◽  
Sara Adobati ◽  
Simona Saporiti ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Amino Acid ◽  
Molecular Dynamics Simulations ◽  
Electrostatic Interactions ◽  
Amino Acid Residues ◽  
Dibasic Amino Acid ◽  
Novel Target ◽  
Function Relationship ◽  
Dynamics Simulations

SLC6A14 (ATB0,+) is a sodium- and chloride-dependent neutral and dibasic amino acid transporter that regulates the distribution of amino acids across cell membranes. The transporter is overexpressed in many human cancers characterized by an increased demand for amino acids; as such, it was recently acknowledged as a novel target for cancer therapy. The knowledge on the molecular mechanism of SLC6A14 transport is still limited, but some elegant studies on related transporters report the involvement of the 12 transmembrane α-helices in the transport mechanism, and describe structural rearrangements mediated by electrostatic interactions with some pivotal gating residues. In the present work, we constructed a SLC6A14 model in outward-facing conformation via homology modeling and used molecular dynamics simulations to predict amino acid residues critical for substrate recognition and translocation. We docked the proteinogenic amino acids and other known substrates in the SLC6A14 binding site to study both gating regions and the exposed residues involved in transport. Interestingly, some of these residues correspond to those previously identified in other LeuT-fold transporters; however, we could also identify a novel relevant residue with such function. For the first time, by combined approaches of molecular docking and molecular dynamics simulations, we highlight the potential role of these residues in neutral amino acid transport. This novel information unravels new aspects of the human SLC6A14 structure–function relationship and may have important outcomes for cancer treatment through the design of novel inhibitors of SLC6A14-mediated transport.

scholarly journals A Novel Cytoplasmic Tail MXXXL Motif Mediates the Internalization of Prostate-specific Membrane Antigen

2003 ◽  
Vol 14 (12) ◽  
pp. 4835-4845 ◽  
Author(s):  
Sigrid A. Rajasekaran ◽  
Gopalakrishnapillai Anilkumar ◽  
Eri Oshima ◽  
James U. Bowie ◽  
He Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Protein ◽  
Interleukin 2 ◽  
Adaptor Protein ◽  
Cytoplasmic Tail ◽  
Prostate Specific Membrane Antigen ◽  
Amino Acid Residues ◽  
Terminal Amino ◽  
Specific Membrane

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the α-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative μ2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.

scholarly journals Structural studies on wheat (Triticum aestivum) proteins lacking phenylalanine and histidine residues

1975 ◽  
Vol 149 (3) ◽  
pp. 725-732 ◽  
Author(s):  
D G Redman
Keyword(s):  
Amino Acids ◽  
Triticum Aestivum ◽  
Amino Acid ◽  
Structural Studies ◽  
Amino Acid Residues ◽  
Sequencing Studies ◽  
Arginine Residues ◽  
Terminal Amino ◽  
Methionine Residues ◽  
High Degree

1. Three very similar proteins, each of approx. 120 amino acid residues but lacking phenylalanine and histidine, were isolated from wheat (Triticum aestivum) flour in sufficient quantities for further structural studies. 2. Each protein, after reduction and carboxymethylation, was cleaved at the three methionine residues with CNBr to give four major peptides, which were isolated. These peptides are suitable for future sequencing studies, as the sums of their amino acid compositions are in good agreement with those of the whole proteins. 3. The N- and C-terminal peptides were identified. 4. Evidence from amino acid analyses, N-terminal amino acids and electrophoretic mobilities of the peptides suggests a high degree of homology between the proteins. Definite differences in C-terminal amino acids and the number of glycine, alanine and arginine residues were found in the C-terminal peptides.

scholarly journals Ornithine decarboxylase stability in HMOA and DH23b cells is not due to post-translational truncation of a C-terminal recognition site

1996 ◽  
Vol 318 (3) ◽  
pp. 879-882
Author(s):  
John L. A. MITCHELL ◽  
Chung-youl CHOE ◽  
Gary G JUDD
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ornithine Decarboxylase ◽  
Cdna Sequence ◽  
Carboxypeptidase Y ◽  
Amino Acid Residues ◽  
Wild Type ◽  
C Terminus ◽  
Variant Cell ◽  
Sds Page

The normally labile ornithine decarboxylase (ODC) becomes unusually stable when Cys-441 is replaced with Trp in the variant cell lines HMOA and DH23b. This stable ODC is also observed to have higher mobility on SDS/PAGE. Because previous studies have shown that ODC stability can be achieved when as few as five amino acid residues are removed from its C-terminus, it was suggested that the amino acid substitution in the variant ODC might alter its conformation sufficiently to promote a similar proteolytic loss of a C-terminal degradation signal, resulting in a stable yet active ODC. To examine this mechanism, amino acids in the C-terminal regions of both wild-type and stable (Trp-441) ODC proteins were released, by means of carboxypeptidase-Y digestion, and identified by HPLC. The C-terminal ends were found to be the same, and are as predicted from the cDNA sequence. This study proves that stability of the Trp-441 form of ODC is not simply due to proteolytic removal of a C-terminal proteasome-targeting sequence, thereby implying that the stabilization of this mutant ODC form must result directly from a conformational change associated with the loss of Cys-441.

INTERFERON SEQUENCE HOMOLOGY AND RECEPTOR BINDING ACTIVITY OF OVINE TROPHOBLAST ANTILUTEOLYTIC PROTEIN

1987 ◽  
Vol 115 (2) ◽  
pp. R13-R15 ◽  
Author(s):  
H.J. Stewart ◽  
S.H.E. McCann ◽  
P.J. Barker ◽  
K.E. Lee ◽  
G.E. Lamming ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Receptor Binding ◽  
Early Pregnancy ◽  
Sequence Homology ◽  
Binding Activity ◽  
Membrane Receptors ◽  
Interferon Α ◽  
Receptor Binding Activity ◽  
Terminal Amino

ABSTRACT Sequencing of the 40 N-terminal amino acids of the blastocyst protein responsible for blocking corpus luteum regression in early pregnancy in sheep revealed a 37% homology with human α-interferon; 28% of the remaining amino acid changes were conservative. 125I-Labelled human α-interferon bound to membrane receptors from sheep uteri with an approximate Kd of 4 × 10−11 M; binding was inhibited by unlabelled α-interferon or purified blastocyst antiluteolytic protein. The blastocyst antiluteolytic protein therefore closely resembles the interferon-α family of antiviral proteins.

scholarly journals Biosynthesis of the Thioquinolobactin Siderophore: an Interesting Variation on Sulfur Transfer

2007 ◽  
Vol 189 (7) ◽  
pp. 2941-2944 ◽  
Author(s):  
Amy M. Godert ◽  
Mi Jin ◽  
Fred W. McLafferty ◽  
Tadhg P. Begley
Keyword(s):  
Amino Acid ◽  
Pseudomonas Fluorescens ◽  
Carrier Protein ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
Sulfur Transfer ◽  
C Terminus ◽  
Interesting Variation ◽  
Terminal Amino

ABSTRACT The thioquinolobactin siderophore from Pseudomonas fluorescens ATCC 17400 utilizes a variation of the sulfur transfer chemistry found in thiamine and molydobterin biosynthesis. A JAMM motif protein cleaves the C-terminal amino acid residues following a diglycine moiety on a small sulfur carrier protein, and the modified C terminus is activated and sulfurylated, forming a thiocarboxylate.

scholarly journals Structural features that make oligopeptides susceptible substrates for hydrolysis by recombinant thimet oligopeptidase

1997 ◽  
Vol 324 (2) ◽  
pp. 517-522 ◽  
Author(s):  
Antonio. C. M CAMARGO ◽  
Marcelo. D GOMES ◽  
Antonia. P REICHL ◽  
Emer. S FERRO ◽  
Saul JACCHIERI ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cleavage Site ◽  
Catalytic Efficiency ◽  
Structural Features ◽  
Amino Acid Residues ◽  
Systematic Analysis ◽  
Peptide Bonds ◽  
C Terminus ◽  
Thimet Oligopeptidase

A systematic analysis of the peptide sequences and lengths of several homologues of bioactive peptides and of a number of quenched-fluorescence (qf) opioid- and bradykinin-related peptides was performed to determine the main features leading the oligopeptides to hydrolysis by the recombinant rat testis thimet oligopeptidase (EC 3.4.24.15). The results indicate that a minimum substrate length of six amino acids is required and that among the oligopeptides six to thirteen amino acid residues long, their susceptibility as substrates is highly variable. Thimet oligopeptidase was able to hydrolyse, with similar catalytic efficiency, peptide bonds having hydrophobic or hydrophilic amino acids as well as proline in the P1 position of peptides, ranging from a minimum of six to a maximum of approximately thirteen amino acid residues. An intriguing observation was the shift of the cleavage site, at a Leu-Arg bond in qf dynorphin-(2–8) [qf-Dyn2–8; Abz-GGFLRRV-EDDnp, where Abz stands for o-aminobenzoyl and EDDnp for N-(2,4-dinitrophenyl) ethylenediamine], to Arg-Arg in qf-Dyn2–8Q, in which Gln was substituted for Val at its C-terminus. Similarly, a cleavage site displacement was also observed with the hydrolysis of the internally quenched-fluorescence bradykinin analogues containing Gln at the C-terminal position, namely Abz-RPPGFSPFR-EDDnp and Abz-GFSPFR-EDDnp are cleaved at the Phe-Ser bond, but Abz-RPPGFSPFRQ-EDDnp and Abz-GFSPFRQ-EDDnp are cleaved at the Pro-Phe bond.

Conformation of branched polypeptides based on poly(L-lysine): The effect of terminal amino acids in the branches

1985 ◽  
Vol 50 (1) ◽  
pp. 228-244 ◽  
Author(s):  
Hana Votavová ◽  
Ferenc Hudecz ◽  
Judit Kajtár ◽  
Jaroslav Šponar ◽  
Karel Bláha ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ionic Strength ◽  
Glutamic Acid ◽  
Amino Acid Residues ◽  
Ordered Structure ◽  
Cd Spectra ◽  
Helix Formation ◽  
Terminal Amino ◽  
Α Helix

CD Spectra of branched polypeptides based on poly(L-lysine) and containing three DL-alanine residues and one to three other L- or D-amino acid residues in the branches were measured in water, water-methanol and water-trifluoroethanol mixtures. In aqueous solutions dependence of the CD spectra on pH and ionic strength was studied. The effect of branch elongation was followed mainly with compounds containing glutamic acid. One terminal D-amino acid residue and also an extension by two L- or D-amino acid residues does not hinder the α-helix formation in the backbone but affects the conditions of its formation. In polypeptides with three L- or D-amino acids additional α-helical segments in the branches are assumed to be formed. For branches with L-amino acids the CD curves express additively the contributions of both helical components, in the case of D-amino acids the increasing population of the ordered structure in branches is manifested by compensation of dichroic contribution of the L-amino acid backbone leading even to enantiomorphous curves.

scholarly journals Covalent Structure and Bioactivity of the Type AII Lantibiotic Salivaricin A2

2017 ◽  
Vol 84 (5) ◽  
Author(s):  
Mengxin Geng ◽  
Frank Austin ◽  
Ronald Shin ◽  
Leif Smith
Keyword(s):  
Amino Acids ◽  
Antibacterial Activity ◽  
Amino Acid ◽  
Amino Acid Residues ◽  
Gram Positive ◽  
Linear Peptide ◽  
Gram Positive Bacteria ◽  
Lipid Ii ◽  
Covalent Structure ◽  
Terminal Amino

ABSTRACTLantibiotics are a class of lanthionine-containing, ribosomally synthesized, and posttranslationally modified peptides (RiPPs) produced by Gram-positive bacteria. Salivaricin A2 belongs to the type AII lantibiotics, which are generally considered to kill Gram-positive bacteria by binding to the cell wall precursor lipid II via a conserved ring A structure. Salivaricin A2 was first reported to be isolated from a probiotic strain,Streptococcus salivariusK12, but the structural and bioactivity characterizations of the antibiotic have remained limited. In this study, salivaricin A2 was purified and its covalent structure was characterized. N-terminal analogues of salivaricin A2 were generated to study the importance for bioactivity of the length and charge of the N-terminal amino acids. Analogue salivaricin A2(3-22) has no antibacterial activity and does not have an antagonistic effect on the native compound. The truncated analogue also lost its ability to bind to lipid II in a thin-layer chromatography (TLC) assay, suggesting that the N-terminal amino acids are important for binding to lipid II. The creation of N-terminal analogues of salivaricin A2 promoted a better understanding of the bioactivity of this antibiotic and further elucidated the structural importance of the N-terminal leader peptide. The antibacterial activity of salivaricin A2 is due not only to the presence of the positively charged N-terminal amino acid residues, but to the length of the N-terminal linear peptide.IMPORTANCEThe amino acid composition of the N-terminal linear peptide of salivaricin A2 is crucial for function. Our study shows that the length of the amino acid residues in the linear peptide is crucial for salivaricin A2 antimicrobial activity. Very few type AII lantibiotic covalent structures have been confirmed. The characterization of the covalent structure of salivaricin A2 provides additional support for the predicted lanthionine and methyl-lanthionine ring formations present in this structural class of lantibiotics. Removal of the N-terminal Lys1 and Arg2 residues from the peptide causes a dramatic shift in the chemical shift values of amino acid residues 7 through 9, suggesting that the N-terminal amino acids contribute to a distinct structural conformer for the linear peptide region. The demonstration that the bioactivity could be partially restored with the substitution of N-terminal alanine residues supports further studies aimed at determining whether new analogues of salivaricin A2 for novel applications can be synthesized.

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