C terminus of NisI provides specificity to nisin

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3543-3549 ◽  
Author(s):  
Timo M. Takala ◽  
Per E. J. Saris
Keyword(s):  
Amino Acids ◽  
Cell Membrane ◽  
Specific Interaction ◽  
Immunity Protein ◽  
C Terminus ◽  
Terminal Amino ◽  
A Site ◽  
Immunity Function ◽  
First Time ◽  
Deletion Series

Nisin-producing Lactococcus lactis protects its own cell membrane against the bacteriocin with the ABC transporter NisFEG, and the immunity lipoprotein NisI. In this study, in order to localize a site for specific nisin interaction in NisI, a C-terminal deletion series of NisI was constructed, and the C-terminally truncated NisI proteins were expressed in L. lactis. The shortest deletion (5 aa) decreased the nisin immunity capacity considerably in the nisin-negative strain MG1614, resulting in approximately 78 % loss of immunity function compared with native NisI. A deletion of 21 aa decreased the immunity level even more, but longer deletions, up to 74 aa, provided the same level of nisin immunity as the 21 aa deletion, i.e. approximately 14 % of the immunity provided by native NisI. Similar to native NisI, all the C-terminally truncated NisI proteins provided higher immunity to nisin in the NisFEG-expressing strain NZ9840 than in MG1614, i.e. approximately 40–50 % of the immunity capacity of native NisI. Then, it was determined whether the NisI C-terminal 21 aa fragment could protect cells against nisin. To target the 21 aa fragment to its natural location, 21 C-terminal amino acids from the subtilin-specific immunity lipoprotein SpaI were replaced by 21 C-terminal amino acids from NisI. The expression of the SpaI′–′NisI fusion in L. lactis strains significantly increased their nisin immunity. This is the first time the immunity function of a lantibiotic immunity protein has been transferred to another protein. However, unlike native NisI, and the C-terminally truncated NisI fragments, the increase in nisin immunity conferred by the SpaI′–′NisI fusion was the same in both the NisFEG strain NZ9840 and MG1614. In conclusion, the SpaI′–′NisI fusion could not enhance nisin immunity by interacting with NisFEG, whereas the C-terminally truncated NisI fragments and native NisI were able to enhance nisin immunity, probably by co-operation with NisFEG. The results made it evident that the C terminus of NisI is involved in specific interaction with nisin, and that it confers specificity for the NisI immunity lipoprotein.

scholarly journals Peptides as antigens. Importance of orientation.

1986 ◽  
Vol 164 (4) ◽  
pp. 1344-1349 ◽  
Author(s):  
T Dyrberg ◽  
M B Oldstone
Keyword(s):  
Peptide Sequence ◽  
Carrier Protein ◽  
Binding Pattern ◽  
Terminal Amino Acid ◽  
C Terminus ◽  
Terminal Amino ◽  
Peptide Characterization ◽  
A Site ◽  
Peptide Antibodies

Factors known to be important in producing protein-reactive peptide antibodies include the accessibility of the region from which the peptide sequence is derived, the hydrophilic-phobic character of the sequence, and the length of the peptide. The data presented here indicate that the orientation of the peptide coupled to a carrier protein also influences the binding pattern of peptide antibodies. An octapeptide, representing a sequence from the alpha chain of the human acetylcholine receptor, was coupled either through an N- or C-terminal cysteine-glycine-glycine linker to a carrier protein and used to immunize rabbits. The resulting antisera reacted at comparable titers to the uncoupled immunizing peptides, but did not crossreact with the identical but opposite-linked peptide. Characterization of the binding to other homologous peptides showed that immunization with the N-terminal-linked peptide induced antibodies reactive specifically with the C-terminal amino acid(s). Immunization with the C-linked peptide resulted in antibodies reactive with a site of the peptide near the C-terminus.

scholarly journals Proteolysis of the Caulobacter McpA Chemoreceptor Is Cell Cycle Regulated by a ClpX-Dependent Pathway

2001 ◽  
Vol 183 (17) ◽  
pp. 5001-5007 ◽  
Author(s):  
Jeng-Wen Tsai ◽  
M. R. K. Alley
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Growth Rate ◽  
Caulobacter Crescentus ◽  
Multicopy Plasmid ◽  
Protein Levels ◽  
C Terminus ◽  
Two Generations ◽  
Terminal Amino ◽  
Dependent Pathway

ABSTRACT Proteolysis is involved in cell differentiation and the progression through the cell cycle in Caulobacter crescentus. We have constitutively expressed the transmembrane chemoreceptor McpA from a multicopy plasmid to demonstrate that McpA degradation is modulated during the cell cycle. The level of McpA protein starts to decrease only when the swarmer cells differentiate into stalked cells. The reduction in McpA protein levels is maintained until the stalked cells develop into predivisional cells, at which point the level returns to that observed in swarmer cells. The cell-cycle-regulated degradation of McpA does not require the last 12 C-terminal amino acids, but it does require three amino acids (AAL) located 15 residues away from the C terminus. The ClpXP protease is essential in C.crescentus for viability, and thus, we tested McpA degradation in xylose conditional mutants. The effect on McpA degradation occurred within two generations from the start of ClpX depletion. The conditional mutants' growth rate was only slightly affected, suggesting that ClpX is directly involved in McpA proteolysis.

scholarly journals The C Terminus of YopT Is Crucial for Activity and the N Terminus Is Crucial for Substrate Binding

2003 ◽  
Vol 71 (8) ◽  
pp. 4623-4632 ◽  
Author(s):  
Isabel Sorg ◽  
Claudia Hoffmann ◽  
Juergen Dumbach ◽  
Klaus Aktories ◽  
Gudula Schmidt
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Cysteine Proteases ◽  
Guanine Nucleotide ◽  
New Family ◽  
C Terminus ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Release Assay ◽  
Terminal Amino ◽  
Inactive Protein

ABSTRACT Recently, it was shown that Yersinia outer protein T (YopT) belongs to a new family of cysteine proteases containing invariant C, H, and D residues that are crucial for its activity. YopT cleaves RhoA, Rac, and Cdc42 at their C termini, thereby releasing them from the membrane. Moreover, YopT inhibits the Rho-rhotekin and Rho-guanine nucleotide dissociation inhibitor interactions. To characterize the active domain of YopT, we constructed N- and C-terminal truncations and expressed them as glutathione S-transferase fusion proteins in Escherichia coli. The toxin fragments were tested for stability by trypsin digestion. The activity of the proteins was studied by membrane release assay, rhotekin pulldown experiments, and microinjection. Whereas deletion of the first 74 N-terminal amino acids did not influence the activity of YopT, deletion of 8 amino acids from the C terminus led to complete loss of activity. N-terminal deletion of 100 amino acids led to an inactive protein, although it still contained the amino acids C139, H258, and D274, which are essential for catalysis. Loss of activity of the N-terminal deletions corresponded to the block of interaction with RhoA, indicating that residues 75 to 100 of YopT are essential for binding to the GTPase. By contrast, when up to 15 amino acids of the C terminus were deleted, the protein had no activity but was still able to interact with RhoA, suggesting a role for the C terminus in the enzyme activity of YopT.

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 The Tip of the Hydrophobic Hairpin of Colicin U Is Dispensable for Colicin U Activity but Is Important for Interaction with the Immunity Protein

1998 ◽  
Vol 180 (16) ◽  
pp. 4111-4115 ◽  
Author(s):  
Holger Pilsl ◽  
David Smajs ◽  
Volkmar Braun
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Membrane ◽  
Published Data ◽  
Hydrophobic Region ◽  
Immunity Protein ◽  
Shigella Boydii ◽  
Transmembrane Segments ◽  
C Terminus ◽  
Colicin B ◽  
Activity Comparison

ABSTRACT The hydrophobic C terminus of pore-forming colicins associates with and inserts into the cytoplasmic membrane and is the target of the respective immunity protein. The hydrophobic region of colicin U ofShigella boydii was mutated to identify determinants responsible for recognition of colicin U by the colicin U immunity protein. Deletion of the tip of the hydrophobic hairpin of colicin U resulted in a fully active colicin that was no longer inactivated by the colicin U immunity protein. Replacement of eight amino acids at the tip of the colicin U hairpin by the corresponding amino acids of the related colicin B resulted in colicin U(575–582ColB), which was inactivated by the colicin U immunity protein to 10% of the level of inactivation of the wild-type colicin U. The colicin B immunity protein inactivated colicin U(575–582ColB) to the same degree. These results indicate that the tip of the hydrophobic hairpin of colicin U and of colicin B mainly determines the interaction with the corresponding immunity proteins and is not required for colicin activity. Comparison of these results with published data suggests that interhelical loops and not membrane helices of pore-forming colicins mainly interact with the cognate immunity proteins and that the loops are located in different regions of the A-type and E1-type colicins. The colicin U immunity protein forms four transmembrane segments in the cytoplasmic membrane, and the N and C termini face the cytoplasm.

scholarly journals Borreliacidal OspC Antibodies Specific for a Highly Conserved Epitope Are Immunodominant in Human Lyme Disease and Do Not Occur in Mice or Hamsters

2005 ◽  
Vol 12 (6) ◽  
pp. 746-751 ◽  
Author(s):  
Steven D. Lovrich ◽  
Dean A. Jobe ◽  
Ronald F. Schell ◽  
Steven M. Callister
Keyword(s):  
Amino Acids ◽  
Lyme Disease ◽  
Surface Protein ◽  
Sensu Stricto ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
C Terminus ◽  
Lyme Disease Vaccine ◽  
Terminal Amino ◽  
Conserved Epitope

ABSTRACT Humans produce highly specific borreliacidal antibodies against outer surface protein C (OspC) shortly after infection with Borrelia burgdorferi sensu stricto. We previously demonstrated the epitope recognized by immunoglobulin M (IgM) and IgG OspC borreliacidal antibodies was located within the 50 amino acids nearest the carboxy (C) terminus. In this study, we show the immunodominant epitope is located in the highly conserved region within the seven C-terminal amino acids. Six early Lyme disease sera that contained borreliacidal activity and IgM and/or IgG OspC antibodies were chosen randomly and adsorbed with truncated OspC containing the 16 or 7 amino acids nearest the C terminus. Adsorptions with each truncated protein abrogated the borreliacidal activity completely. In addition, only small concentrations of OspC antibodies remained detectable by enzyme-linked immunosorbent assay and Western blotting. Moreover, borreliacidal OspC antibodies were not induced in laboratory mice or hamsters despite heavy infections with B. burgdorferi spirochetes. These findings confirm that borreliacidal antibodies comprise the majority of the IgM and IgG OspC antibody response in human Lyme disease and that the epitope is located in the highly conserved C terminus. In addition, rodent animal models appear to be inappropriate subjects for assessing the effectiveness of the epitope for serodiagnosis or as a human Lyme disease vaccine.

Truncated atrial natriuretic factor analogs retain full agonist activity

1991 ◽  
Vol 69 (10) ◽  
pp. 1622-1627 ◽  
Author(s):  
W. H. Holleman ◽  
G. P. Budzik ◽  
E. M. Devine ◽  
D. M. Pollock ◽  
T. J. Opgenorth ◽  
...  
Keyword(s):  
Amino Acids ◽  
Atrial Natriuretic Factor ◽  
Cyclic Peptides ◽  
Hypotensive Activity ◽  
Agonist Activity ◽  
Natriuretic Factor ◽  
C Terminus ◽  
Terminal Amino ◽  
Atrial Natriuretic

The synthesis, receptor binding, and agonist activity of a series of truncated atrial natriuretic analogs (ANF) are described. These analogs incorporate two portions of the native 28 amino peptide, the eight amino acids C-terminal to Cys7, and two amino acids from the C-terminus (phenylalanine and arginine), into disulfide-bonded cyclic peptides. The inclusion of the C-terminal amino acids converted the ANF analogs from receptor ligands to full agonists, as measured by several methods, including the stimulation of cGMP biosynthesis in endothelial cells, inhibition of aldosterone biosynthesis in rat adrenal cells, and natriuretic–hypotensive activity in vivo. The most potent analogs have cyclohexylalanine (Cha) at position 8. The lead compound (Arg6,Cha8 ANF 6–15 Phe-Arg-Cys-NH2) is a tridecapeptide that integrates the C-terminal amino acids inside the disulfide ring. This peptide, designated as A-68828, has a binding affinity of IC50 = 120 nM, approximately 1/400 of ANF 1–28. However, this analog, in vivo, is only slightly less natriuretic (1/20–1/50) than ANF 1–28. Unlike the native peptide, A-68828 is only mildly hypotensive and at the highest concentration tested reduced blood pressure less than 15 mmHg (1 mmHg = 133.322 Pa). A-68828 inhibited ACTH-induced aldosterone release to a greater extent than ANF 1–28: 100 vs. 50%. The selective natriuretic activity of A-66828, relative to ANF, suggests clinical utility for the treatment of acute renal failure.Key words: atrial natriuretic factor, renal failure, dopamine, peptide analogs, cGMP.

scholarly journals Two Distinct Domains within CIITA Mediate Self-Association: Involvement of the GTP-Binding and Leucine-Rich Repeat Domains

2001 ◽  
Vol 21 (9) ◽  
pp. 3001-3011 ◽  
Author(s):  
Michael W. Linhoff ◽  
Jonathan A. Harton ◽  
Drew E. Cressman ◽  
Brian K. Martin ◽  
Jenny Pan-Yun Ting
Keyword(s):  
Amino Acids ◽  
Major Histocompatibility Complex Gene ◽  
Binding Domain ◽  
Signaling Proteins ◽  
Leucine Rich Repeat ◽  
Histocompatibility Complex ◽  
Gtp Binding ◽  
C Terminus ◽  
Self Association ◽  
First Time

ABSTRACT CIITA is the master regulator of class II major histocompatibility complex gene expression. We present evidence that CIITA can self-associate via two domains: the C terminus (amino acids 700 to 1130) and the GTP-binding domain (amino acids 336 to 702). Heterotypic and homotypic interactions are observed between these two regions. Deletions within the GTP-binding domain that reduce GTP-binding and transactivation function also reduce self-association. In addition, two leucine residues in the C-terminal leucine-rich repeat region are critical for self-association as well as function. This study reveals for the first time a complex pattern of CIITA self-association. These interactions are discussed with regard to the apoptosis signaling proteins, Apaf-1 and Nod1, which share domain arrangements similar to those of CIITA.

A basic region neighboring the lysine-rich C-terminus of protein SRP19 is required for binding to signal recognition particle RNA

1991 ◽  
Vol 69 (9) ◽  
pp. 649-654 ◽  
Author(s):  
Christian Zwieb
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Signal Recognition Particle ◽  
Site Directed Mutagenesis ◽  
Signal Recognition ◽  
C Terminus ◽  
Terminal Amino ◽  
Rna Complexes ◽  
Mutant Polypeptides

To identify some of the determinants in the 19-kilodalton protein of signal recognition particle (SRP19) for binding to signal recognition particle RNA, two mutant derivatives of the SRP19 were constructed, lacking 14 and 24 C-terminal amino acids. Polypeptides were transcribed and translated in vitro and tested for their ability to bind to signal recognition particle RNA by retention of protein–RNA complexes on DEAE–Sepharose. Both mutant polypeptides form complexes with the RNA, demonstrating that the 24 C-terminal amino acids, which include a lysine-rich sequence at positions 136–144, are dispensable. A third mutant polypeptide, in which eight additional amino acids were removed by oligonucleotide-directed digestion of the mRNA, was unable to bind. The amino acids in the sequence PKLKTRTQ correspond to positions 113–120; they are suggested to be involved in interaction with signal recognition particle RNA.Key words: signal recognition particle, site-directed mutagenesis, protein–RNA binding.

The AtMYB12 activation domain maps to a short C-terminal region of the transcription factor

2017 ◽  
Vol 72 (7-8) ◽  
pp. 251-257 ◽  
Author(s):  
Ralf Stracke ◽  
Neslihan Turgut-Kara ◽  
Bernd Weisshaar
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Myb Transcription Factor ◽  
Activation Domain ◽  
Domain Swap ◽  
Terminal Amino ◽  
R2r3 Myb ◽  
Target Promoters ◽  
Activation Capacity ◽  
Deletion Series

AbstractTheArabidopsis thalianaR2R3-MYB transcription factor MYB12 is a light-inducible, flavonol-specific activator of flavonoid biosynthesis. The transactivation activity of theAtMYB12 protein was analyzed using a C-terminal deletion series in a transientA. thalianaprotoplast assay with the goal of mapping the activation domain (AD). Although the deletion of the last 46 C-terminal amino acids did not affect the activation capacity, the deletion of the last 98 amino acids almost totally abolished transactivation of two different target promoters. A domain swap experiment using the yeast GAL4 DNA-binding domain revealed that the region from positions 282 to 328 ofAtMYB12 was sufficient for transactivation. In contrast to the R2R3-MYB ADs known thus far, that ofAtMYB12 is not located at the rearmost C-terminal end of the protein. TheAtMYB12 AD is conserved in other experimentally proven R2R3-MYB flavonol regulators from different species.

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