Current Protein and Peptide Science, Vol. 5, No. 5, 2004
Contents
Circular Peptides and
Proteins
Guest Editor: David J. Craik
Discovery, Structure and Biological
Activities of the Cyclotides Pp.297-315
David. J. Craik, Norelle L. Daly, Jason
Mulvenna, Manuel R. Plan and Manuela Trabi
Novel Strategies for Isolation and
Characterization of Cyclotides: The Discovery of Bioactive Macrocyclic Plant
Polypeptides in the Violaceae Pp.317-329
Ulf Goransson, Erika Svangard, Per Claeson
and Lars Bohlin
Anti-HIV Cyclotides Pp.331-340
Kirk R. Gustafson, Tawnya C. McKee and Heidi
R. Bokesch
Squash Inhibitors: From Structural Motifs to
Macrocyclic Knottins Pp.341-349
Laurent Chiche, Annie Heitz, Jean-Christophe
Gelly, Jerome Gracy, Pham T.T. Chau, Phan T. Ha, Jean-Francois Hernandez and
Dung Le-Nguyen
Sunflower Trypsin Inhibitor-1 Pp.351-364
Michael L.J. Korsinczky, Horst Joachim
Schirra and David J. Craik
q-Defensins: Cyclic Antimicrobial Peptides
Produced by Binary Ligation of Truncated a-Defensins
Pp.365-371
Michael E. Selsted
Retrocyclins: Using Past as Prologue Pp.373-381
Microcin J25, from the Macrocyclic to the
Lasso Structure: Implications for Biosynthetic, Evolutionary and
Biotechnological Perspectives Pp.383-391
Sylvie Rebuffat, Alain Blond, Delphine
Destoumieux-Garzon, Christophe Goulard and Jean Peduzzi
The Circular Bacteriocins Gassericin A and Circularin
A Pp.393-398
Yasushi Kawai, Rober Kemperman, Jan Kok and Tadao Saito
Peptide AS-48: Prototype of a New Class of
Cyclic Bacteriocins Pp.399-416
Mercedes Maqueda, Antonio Galvez, Manuel Martinez Bueno, Maria Jose
Sanchez-Barrena, Carlos Gonzalez, Armando Albert, Manuel Rico and Eva Valdivia
Protein Circlets as Sex Pilus Subunits Pp.417-424
Markus Kalkum, Ralf Eisenbrandt and Erich Lanka
Abstracts
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Discovery,
Structure and Biological Activities of the Cyclotides
David. J. Craik, Norelle L. Daly, Jason
Mulvenna, Manuel R. Plan and Manuela Trabi
The cyclotides are a family of small disulfide rich proteins that have a cyclic peptide backbone and a cystine knot formed by three conserved disulfide bonds. The combination of these two structural motifs contributes to the exceptional chemical, thermal and enzymatic stability of the cyclotides, which retain bioactivity after boiling. They were initially discovered based on native medicine or screening studies associated with some of their various activities, which include uterotonic action, anti-HIV activity, neurotensin antagonism, and cytotoxicity. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaceae families and their natural function in plants appears to be in host defense: they have potent activity against certain insect pests and they also have antimicrobial activity. There are currently around 50 published sequences of cyclotides and their rate of discovery has been increasing over recent years. Ultimately the family may comprise thousands of members. This article describes the background to the discovery of the cyclotides, their structural characterization, chemical synthesis, genetic origin, biological activities and potential applications in the pharmaceutical and agricultural industries. Their unique topological features make them interesting from a protein folding perspective. Because of their highly stable peptide framework they might make useful templates in drug design programs, and their insecticidal activity opens the possibility of applications in crop protection.
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Novel Strategies
for Isolation and Characterization of Cyclotides: The Discovery of Bioactive
Macrocyclic Plant Polypeptides in the Violaceae
Ulf Goransson, Erika Svangard, Per Claeson
and Lars Bohlin
This review focuses on the discovery of cyclotides in the plant family Violaceae, their isolation and their anticancer effects. These macrocyclic plant peptides consist of about 30 amino acids, including three conserved disulfide bonds in a cystine knotted arrangement, which renders them a remarkable stability. Their unique structure, combined with a wide array of biological activities, makes them of great interest as possible leads in drug development or as carriers of grafted peptide sequences. Here we describe the work conducted in our laboratory, which started with the overall aim of identifying peptides and small proteins of the size 10-50 amino acid residues in plants with novel chemical structures and biological profiles with a potential for drug development or for use as pharmacological tools. Thus we developed a fractionation protocol to directly address major challenges encountered when dealing with plant material, such as removal of chlorophyll, polyphenols, and low molecular compounds omnipresent in plants. Using this protocol, we then discovered a suite of cyclotides, the varv peptides, from the plant Viola arvensis (Violaceae). Following this, separation methods directly targeting cyclotides were developed, e.g. by adsorption, ion exchange chromatography and solventsolvent partitioning, which then were used in the isolation of additional cyclotides. To structurally examine cyclotides we have also developed methods based on mass spectrometry for cyclotide sequencing and mapping of disulfide bonds. Finally, to assess structure-activity relationships, regarding their anti-cancer and cytotoxic effects that we focus upon, we have also characterized the three dimensional structure of cyclotides by homology modeling techniques.
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Anti-HIV
Cyclotides
Kirk R. Gustafson, Tawnya C. McKee and Heidi
R. Bokesch
The cyclotides are a recently discovered, structurally unique family of bioactive plant peptides. Their discovery spawned a series of structural analyses, synthetic efforts, and studies to define the biosynthesis and biological properties of these novel peptide metabolites. Cyclotides have a head-to-tail cyclized amino acid backbone and a conserved cystine knot motif that provides an extremely stable structural framework. They all share a common global fold and are highly resistant to denaturation and to cleavage by proteolytic enzymes. However, these macrocyclic peptides are quite permissive to amino acid substitutions or additions in several peripheral loop regions, since changes in these loops do not alter the core cyclotide structure. These features make cyclotides attractive templates for incorporating desired amino acid sequences and then delivering these peptide sequences in a well defined, highly stable framework. Cyclotides likely function in a defensive role in the source plants since they exhibit a broad spectrum of antimicrobial activity and are detrimental to the growth and survival of herbivorous insects. Cyclotides are gene-encoded polypeptides that are cleaved from larger precursor proteins and then cyclized. This review summarizes research done on a subset of cyclotides that were discovered due to their HIV inhibitory properties. It details the isolation and characterization of these compounds and describes this work in the context of our current state of knowledge of the entire cyclotide family.
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Squash Inhibitors:
From Structural Motifs to Macrocyclic Knottins
Laurent Chiche, Annie Heitz, Jean-Christophe
Gelly, Jerome Gracy, Pham T.T. Chau, Phan T. Ha, Jean-Francois Hernandez and
Dung Le-Nguyen
In this article, we will first introduce the squash inhibitors, a well established family of highly potent canonical serine proteinase inhibitors isolated from Cucurbitaceae. The squash inhibitors were among the first discovered proteins with the typical knottin fold shared by numerous peptides extracted from plants, animals and fungi. Knottins contain three knotted disulfide bridges, two of them arranged as a Cystine-Stabilized Beta-sheet motif.
In contrast to cyclotides for which no natural linear homolog is known, most squash inhibitors are linear. However, Momordica cochinchinensis Trypsin Inhibitor-I and -II (MCoTI-I and -II), 34-residue squash inhibitors isolated from seeds of a common Cucurbitaceae from Vietnam, were recently shown to be macrocyclic. In these circular squash inhibitors, a short peptide linker connects residues that correspond to the N- and C-termini in homologous linear squash inhibitors.
In this review we present the isolation, characterization, chemical synthesis, and activity of these macrocyclic knottins. The solution structure of MCoTI-II will be compared with topologically similar cyclotides, homologous linear squash inhibitors and other knottins, and potential applications of such scaffolds will be briefly discussed.
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Sunflower
Trypsin Inhibitor-1
Michael L.J. Korsinczky, Horst Joachim
Schirra and David J. Craik
SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar Ki value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors.
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q-Defensins:
Cyclic Antimicrobial Peptides Produced by Binary Ligation of Truncated a-Defensins
Michael E. Selsted
The first cyclic peptide discovered in animals is an antimicrobial octadecapeptide that is expressed in leukocytes of rhesus monkeys. The peptide, termed rhesus q-defensin 1 (RTD-1) is the prototype of a new family of antimicrobial peptides, which like the previously characterized a- and b-defensin families, possesses broad spectrum microbicidal activities against bacteria, fungi, and protects mononuclear cells from infection by HIV-1. The cyclic q-defensin structure is essential for a number of its antimicrobial properties, as demonstrated by the markedly reduced microbicidal activities of de-cyclized q-defensin analogs. Genetic and biochemical experiments disclosed that the biosynthesis of RTD-1 results from the head-to-tail joining of two nine-amino acid peptides, each of which is donated by a separate precursor polypeptide, which are in fact C-terminally truncated pro-a-defensins. Alternate combinations of the two nonapeptides generate two additional macaque q-defensins, RTD-2 and RTD-3. Humans do not express q-defensin peptides, but mRNAs encoding at least two q-defensins are expressed in human bone marrow. However, in each case the open reading frame is interrupted by a stop codon in the signal peptide-coding region. The mature q-defensin peptide is a two-stranded b-sheet that, like the a- and b-defensins, is stabilized by three disulfides. However, the parallel orientation of the q- defensin disulfide arrangement allows for substantial flexibility around its short axis. Unlike a- and b-defensins, RTD-1 lacks an amphiphilic topology. This may partially explain the unusual interaction between q-defensins and phospholipid bilayers.
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Retrocyclins:
Using Past as Prologue
Retrocyclins are synthetic q-defensins that were reconstructed from genetic blueprints that had remained unused for at least 7.5 million years. From phylogenetic studies, it appears that q-defensins arose in Old World Monkeys, after that lineage had separated from the prosimians and New World Monkeys. Although some nonhuman primates continue to produce q-defensin peptides today, Homo sapiens and his gorilla, bonobo, and chimpanzee relatives do not. Their inability to do so reflects the common inheritance of defective q-defensin genes which contain a premature stop codon that aborts translation. We can only speculate if this ancient genetic event has any contemporary relevance. However, it is noteworthy that synthetic retrocyclins can prevent the entry of HIV-1 and other viruses (e.g., HSV-2) into otherwise susceptible human target cells. Our studies suggest that their antiviral properties are intimately linked to an ability to bind carbohydrate epitopes displayed by viral and cell-surface glycoproteins involved in viral entry. The ability of retrocyclins to recognize and bind carbohydrate and glycan moieties is shared by q-defensins (RTDs) from rhesus monkeys and by several – but not all – human a-defensins. In addition to being the only cyclic peptides of animal origin, the lectin-like activity of q-defensins gives them the added distinction of being the smallest sugar-binding molecules of natural origin identified to date. This unusual combination makes q-defensins intriguing molecular prototypes that could be used to design novel carbohydrate-binding or antiviral agents.
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Microcin J25, from the Macrocyclic to the
Lasso Structure: Implications for Biosynthetic, Evolutionary and
Biotechnological Perspectives
Sylvie Rebuffat, Alain Blond, Delphine
Destoumieux-Garzon, Christophe Goulard and Jean Peduzzi
Microcin J25 (MccJ25) is a cyclic antibacterial peptide secreted by a fecal isolate of Escherichia coli. It exerts highly potent activity on Salmonella and Escherichia species. The microcin is recognized at the outer membrane of sensitive strains by the FhuA multifunctional protein, which belongs to the iron/siderophore receptor family, and inhibits bacterial transcription through binding to the RNA-polymerase b' subunit. The mcjABCD genetic system carried by the wild type 50-kb pTUC100 plasmid contains four genes involved in MccJ25 production and immunity. MccJ25 results from the proteolytic cleavage of a 58-residue precursor at a specific Lys-Gly bond. The resulting mature peptide consists of 21 unmodified amino acids, mostly hydrophobic and includes a single dehydration. The initially described macrocyclic structure of MccJ25, which mostly relied on manual Edman sequencing of the thermolysin-cleaved form (t-MccJ25), involved a head-to-tail cyclisation of the 21-residue precursor. This structure did not prove to be consistent with recent ITMS CID experiments conducted either on the native microcin or on peptides resulting from acidic or enzymatic cleavages, which are in favour of an 8-residue ring followed by a 13-residue tail. Cyclisation thus occurs between the N-terminus (Gly1) and the Glu8 side chain carboxyl group. The solution three-dimensional structure shows threading of the tail into the ring, thus forming a highly stable lasso type structure. Such a structure was described previously for enzyme inhibitors from Actinobacteria and is consistent with the ability of MccJ25 to inhibit RNA polymerase. The lasso structure is discussed in terms of phylogenetical and biotechnological perspectives.
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The Circular Bacteriocins Gassericin A and
Circularin A
Yasushi Kawai, Rober Kemperman, Jan Kok and
Tadao Saito
Gassericin A, a bacteriocin produced by Lactobacillus gasseri LA39, shows antibacterial activity against a number of Gram-positive food-borne pathogenic bacteria. Circularin A produced by Clostridium beijerinckii ATCC25752 is active against C. tyrobutyricum, a known cheese-spoilage bacterium. Both bacteriocins were purified to homogeneity from culture supernatants by reverse-phase chromatography and the subsequently determined amino acid sequences were used to clone the bacteriocin structural genes. Mature gassericin A and circularin A are class V circular bacteriocins comprised of 58 and 69 amino acid residues, respectively. Both bacteriocins are resistant to several peptidases and proteases, as are other cyclic bacteriocins. Heterologous expression of gassericin A in Escherichia coli was used to produce a non-cyclic mature peptide, which was shown to have a specific activity 173-fold lower than the circular molecule. The minimal region for production and secretion of active circularin A is comprised of five genes, as was deduced by heterologous gene expression in Enterococcus faecalis. Gassericin A and circularin A have limited mutual similarity in their primary sequences. Unlike most bacteriocins, including gassericin A, circularin A has a three-aminoacid-leader sequence.
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Peptide AS-48: Prototype of a New Class of
Cyclic Bacteriocins
Mercedes Maqueda, Antonio Galvez, Manuel
Martinez Bueno, Maria Jose Sanchez-Barrena, Carlos Gonzalez, Armando Albert,
Manuel Rico and Eva Valdivia
After the discovery of bacteriocin AS-48, a 70-residue cyclic peptide produced by Enterococcus faecalis subsp. liquefaciens, some naturally-occurring cyclic proteins from bacteria have been reported. AS-48 is encoded by the 68-kb pheromone-responsive plasmid pMB2, and the gene cluster involved in production and immunity has been identified and sequenced. This peptide exerts a bactericidal action on sensitive cells (most of the Gram-positive and some Gram-negative bacteria). Its target is the cytoplasmic membrane, in which it opens pores, leading to the dissipation of the proton motive force and cell death, a mechanism similar to that proposed for the action of defensins or, most generally, cationic antibacterial peptides. This fact, together with its remarkable stability and solubility over a wide pH range, suggest that this bacteriocin could be a good candidate as a natural food preservative. The amino acid composition of purified AS-48 shows the absence of modified or dehydrated residues, making it clearly different from lantibiotics. Bacteriocin AS-48 also differs from defensins in that it does not contain cysteines and consequently no disulfide bridges, which makes is high stability even more remarkable. Composition analysis of AS-48 shows a high proportion of basic to acidic amino acids, conferring to this peptide a strong basic character, with an isoelectric point close to 10.5. Determination of the AS- 48 structural gene DNA sequence, together with the sequences of AS-48 protease digestion fragments and mass spectrometry determinations, allowed us to determine unambiguously the cyclic structure of the molecule, being the first example of a posttranslational modification in which a cyclic structure arises from a ‘‘head-to-tail’’ linkage. We have solved the three-dimensional structure of AS-48 in solution, and it consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. Interestingly, the head-to-tail peptide link between Trp-70 and Met-1 lies in the middle of alpha-helix 5, which is shown to have a pronounced effect on the stability of the three-dimensional structure. Analysis of structure-function relationship allowed us to propose models to understand the aspects of the molecular function of AS-48. The purpose of this work is to review recent developments in our understanding about the biochemical and biological characteristics and structure of this unusual type of bacteriocin.
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Protein Circlets as Sex Pilus Subunits
Markus Kalkum, Ralf Eisenbrandt and Erich
Lanka
The largest circular protein structures discovered define a class of transfer proteins acting in bacterial conjugation and type IV secretion. Proteins ranging from 73 to 78 residues with head-to-tail peptide bonds constitute the major subunit of conjugative pili of some type IV secretion systems. Their plasmid-encoded precursors are enzymatically processed and cyclized before being assembled into pili. These extra-cellular surface filaments mediate physical contact between donor and recipient cell or pathogen and host cell. Pili are essential prerequisites for DNA and protein transfer. A membrane-bound signal peptidase-like enzyme is responsible for the circularization reaction. Site-directed mutagenesis and mass spectrometry has been used extensively to unravel the mechanism of the enzyme-substrate interaction of the pilin maturation process.