Manuela Trabi

Circular proteins: no end in sight

Circular proteins are a recently discovered phenomenon. They presumably evolved to confer advantages over ancestral linear proteins while maintaining the intrinsic biological functions of those proteins. In general, these advantages include a reduced sensitivity to proteolytic cleavage and enhanced stability. In one remarkable family of circular proteins, the cyclotides, the cyclic backbone is additionally braced by a knotted arrangement of disulfide bonds that confers additional stability and topological complexity upon the family. This article describes the discovery, structure, function and biosynthesis of the currently known circular proteins. The discovery of naturally occurring circular proteins in the past few years has been complemented by new chemical and biochemical methods to make synthetic circular proteins; these are also briefly described.

Distribution and evolution of circular miniproteins in flowering plants

Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed >200 Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed >140 species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the divergence of Asterids and Rosids (∼125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.

Tissue-Specific Expression of Head-to-Tail Cyclized Miniproteins in Violaceae and Structure Determination of the Root Cyclotide Viola hederacea root cyclotide1

The plant cyclotides are a family of 28 to 37 amino acid miniproteins characterized by their head-to-tail cyclized peptide backbone and six absolutely conserved Cys residues arranged in a cystine knot motif: two disulfide bonds and the connecting backbone segments form a loop that is penetrated by the third disulfide bond. This knotted disulfide arrangement, together with the cyclic peptide backbone, renders the cyclotides extremely stable against enzymatic digest as well as thermal degradation, making them interesting targets for both pharmaceutical and agrochemical applications. We have examined the expression patterns of these fascinating peptides in various Viola species (Violaceae). All tissue types examined contained complex mixtures of cyclotides, with individual profiles differing significantly. We provide evidence for at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Furthermore, we have isolated one cyclotide expressed only in underground parts of V. hederacea and characterized its primary and three-dimensional structure. We propose that cyclotides constitute a new family of plant defense peptides, which might constitute an even larger and, in their biological function, more diverse family than the well-known plant defensins.

A Comparison of the Self-association Behavior of the Plant Cyclotides Kalata B1 and Kalata B2 via Analytical Ultracentrifugation

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted. We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mm, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe6, and the second involving a charge-charge interaction with the Asp25 residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

Structures of naturally occurring circular proteins from bacteria

Bacteria produce a wide variety of both proteinaceous and nonproteinaceous molecules for defense, mediation of microbial competitions, and signaling purposes. Of the protein-based molecules, there are examples of large folded polypeptides that are translated conventionally from genes as well as

Crystal structure of textilinin-1, a Kunitz-type serine protease inhibitor from the venom of the Australian common brown snake (Pseudonaja textilis)

Textilinin‐1 is a Kunitz‐type serine protease inhibitor isolated from the venom of the Australian common brown snake, Pseudonaja textilis. This molecule binds to and blocks the activity of a range of serine proteases, including plasmin and trypsin. Textilinin‐1’s ability to inhibit plasmin, a protease involved in fibrinolysis, has raised the possibility that it could be used as an alternative to aprotinin (Trasylol) as a systemic antibleeding agent in surgery. Here, the crystal structure of free recombinant textilinin‐1 has been determined to 1.63 Å, with three molecules observed in the asymmetric unit. All of these have a similar overall fold to aprotinin, except that the canonical loop for one of the molecules is inverted such that the side chain of the P1′ residue, Val18, is partially buried by intramolecular contacts to Pro15, Thr13, and Ile36. In aprotinin, the P1′ residue is Ala16, whose side chain is too small to form similar contacts. The loop inversion in textilinin‐1 is facilitated by changes in backbone dihedral angles for the P1 and P2′ residues, such that they alternate between values in the β‐sheet and α‐helical regions of the Ramachandran plot. In a comparison with the structures of all other known Kunitz‐type serine protease inhibitors, no such conformational variability has been observed. The presence of the bulkier valine as the P1′ residue in textilinin‐1 appears to be a major contributor to reducing the binding affinity for plasmin as compared to aprotinin (3.5 nm versus 0.053 nm) and could also account for an observed narrower binding specificity.

Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss.

Aprotinin has been used widely in surgery as an anti‐bleeding agent but is associated with a number of side effects. We report that textilinin‐1, a serine protease inhibitor from Pseudonaja textilis venom with sequence relatedness to aprotinin, is a potent but reversible plasmin inhibitor and has a narrower range of protease inhibition compared to aprotinin. Like aprotinin, textilinin‐1 at 5 μmol/l gave almost complete inhibition of tissue plasminogen activator‐induced fibrinolysis of whole blood clots. The activated partial thromboplastin time for plasma was markedly increased by aprotinin but unaffected by textilinin‐1. In a mouse tail‐vein bleeding model, intravenous textilinin‐1 and aprotinin caused similar decreases in blood loss but time to haemostasis in the textilinin‐treated animals was significantly shorter than in aprotinin‐treated mice. Based on these data, textilinin‐1 merits further investigation as a therapeutic alternative to aprotinin.

Circular proteins from Melicytus (Violaceae) refine the conserved protein and gene architecture of cyclotides

Cyclotides are cyclic disulfide rich mini-proteins found in various Rubiaceae (coffee family), Violaceae (violet family) and Cucurbitaceae (squash family) plant species. Within the Violaceae, cyclotides have been found in numerous species of the genus Viola as well as species from two other genera, namely Hybanthus and Leonia. This is the first in-depth report of cyclotides in the genus Melicytus (Violaceae). We present the chromatographic profiles of extracts of eight Melicytus species and one Melicytus hybrid that were found to contain these circular peptides. We isolated and characterised five novel cyclotides (mra1 to mra5) from the aerial parts of a common New Zealand tree, Melicytus ramiflorus. All five peptides show the characteristics of the bracelet subfamily of cyclotides. Furthermore, we isolated 17 non-redundant cDNA clones from the leaves of Melicytus ramiflorus encoding cyclotide prepropeptides. This detailed report on the presence of cyclotides in several species of the genus Melicytus further strengthens our hypothesis that cyclotides are ubiquitous in Violaceae family plants and provides additional insight into the biochemical processing mechanisms that produce the cyclic protein backbone of this unique family of ultra-stable plant proteins.

Comparison of Textilinin-1 with Aprotinin as Serine Protease Inhibitors and as Antifibrinolytic Agents

Textilinin-1 (Q8008) was isolated from the venom of the Pseudonaja textilis and has a 47% sequence identity to the antihaemorrhagic therapeutic agent aprotinin. When equimolar concentrations of enzyme and aprotinin were pre-incubated, plasmin was inhibited 100%, plasma kallikrein 58%, and tissue kallikrein 99%. Under the same conditions, textilinin-1 inhibited plasmin 98%, plasma kallikrein 16% and tissue kallikrein 17%. Whole blood clot lysis was inhibited strongly by both aprotinin and textilinin-1, as shown by thrombelastography. At 2 µM inhibitor lysis initiated by t-PA was greater than 99% inhibited by aprotinin (LY60 = 0.4 ± 0.1) whereas textilinin-1, inhibited lysis by 91% (LY60 = 8.9 ± 0.7). The same trend was found with the lysis of euglobulin fractions. From these data textilinin-1 appears to be a more specific plasmin inhibitor than aprotinin but aprotinin inhibits clot lysis to a greater extent.

The cyclic antimicrobial peptide RTD-1 induces stabilized lipid-peptide domains more efficiently than its open-chain analogue

The effects of a mammalian cyclic antimicrobial peptide, rhesus theta defensin 1 (RTD‐1) and its open chain analogue (oRTD‐1), on the phase behaviour and structure of model membrane systems (dipalmitoyl phosphatidylcholine, DPPC and dipalmitoyl phosphatidylglycerol, DPPG) were studied. The increased selectivity of RTD‐1 for anionic DPPG over zwitterionic DPPC was shown by differential scanning calorimetry. RTD‐1, at a molar peptide–lipid ratio of 1:100, induced considerable changes in the phase behaviour of DPPG, but not of DPPC. The main transition temperature, T m, was unchanged, but additional phase transitions appeared above T m. oRTD‐1 induced similar effects. However, the effects were not observable below a peptide:lipid molar ratio of 1:50, which correlates with the weaker biological activity of oRTD‐1. Small‐ and wide‐angle X‐ray scattering revealed for DPPG the appearance of additional structural features induced by RTD‐1 above T m, which were interpreted as correlated lamellar structures, with increased order of the fatty acyl side chains of the lipid. It is proposed that after initial electrostatic interaction of the cationic rim of the peptide with the anionic DPPG headgroups, leading to stabilized lipid–peptide clusters, the hydrophobic face of the peptide assists in its interaction with the fatty acyl side chains eventually leading to membrane disruption.