Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.

Structure of Petunia hybrida Defensin 1, a Novel Plant Defensin with Five Disulfide Bonds

The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by H-1 NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first example of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.

Microcin J25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone

Microcin J25 is a 21 amino acid bacterial peptide that has potent antibacterial activity against Gram-negative bacteria, resulting from its interaction with RNA polymerase. The peptide was previously proposed to have a head-to-tail cyclized peptide backbone and a tight globular structure (Blond, A., Peduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthelemy, M., Prigent, Y., Salomon, R. A., Farias, R. N., Moreno, F. & Rebuffat, S. Eur. J. Biochem. 1999, 259, 747-755). It exhibits remarkable thermal stability for a peptide of its size lacking disulfide bonds and in part this was previously proposed to derive from its macrocyclic structure. We show here that in fact the peptide does not have a head-to-tail cyclic structure but rather a side chain to backbone cyclization between Glu8 and the N-terminus. This creates an embedded ring that is threaded by the C-terminal tail of the molecule, forming a noose-like feature. The three-dimensional structure deduced from NMR data suggests that slippage of the noose is prevented by two aromatic residues flanking the embedded ring. Unthreading does not occur even when the molecule is enzymatically digested with thermolysin. The new structural interpretation fully accounts for previously reported NMR and biophysical data and is consistent with the remarkable stability of this potent antimicrobial peptide.

Twists, knots, and rings in proteins: Structural definition of the cyclotide framework

In recent years an increasing number of miniproteins containing an amide-cyclized backbone have been discovered. The cyclotide family is the largest group of such proteins and is characterized by a circular protein backbone and six conserved cysteine residues linked by disulfide bonds in a tight core of the molecule. These form a cystine knot in which an embedded ring formed by two of the disulfide bonds and the connecting backbone segment is threaded by a third disulfide bond. In the current study we have undertaken high resolution structural analysis of two prototypic cyclotides, kalata B1 and cycloviolacin O1, to define the role of the conserved residues in the sequence. We provide the first comprehensive analysis of the topological features in this unique family of proteins, namely rings (a circular backbone), twists (a cis-peptide bond in the Mobius cyclotides) and knots (a knotted arrangement of the disulfide bonds).

Solution structure of the cyclotide palicourein: Implications for the development of a pharmaceutical framework

The cyclotides are a family of disulfide-rich proteins from plants. They have the characteristic structural features of a circular protein backbone and a knotted arrangement of disulfide bonds. Structural and biochemical studies of the cyclotides suggest that their unique physiological stability can be loaned to bioactive peptide fragments for pharmaceutical and agricultural development. In particular, the cyclotides incorporate a number of solvent-exposed loops that are potentially suitable for epitope grafting applications. Here, we determine the structure of the largest known cyclotide, palicourein, which has an atypical size and composition within one of the surface-exposed loops. The structural data show that an increase in size of a palicourein loop does not perturb the core fold, to which the thermodynamic and chemical stability has been attributed. The cyclotide core fold, thus, can in principle be used as a framework for the development of useful pharmaceutical and agricultural bioactivities.

Metal Clips Induce Folding of a Short Unstructured Peptide into an a-Helix via Turn Conformations in Water. Kinetic versus Thermodynamic Products

Short peptides corresponding to two to four a-helical turns of proteins are not thermodynamically stable helices in water. Unstructured octapeptide Ac-His1*-Ala2-Ala3-His4*-His5*-Glu6-Leu7-His8*-NH2 (1) reacts with two [Pd ((NH2)-N-15(CH2)(2) (NH2)-N-15)(NO3)(2)] in water to form a kinetically stable intermediate, [{Pden}(2)-{(1,4)(5,8)-peptide}](2), in which two 19-membered metallocyclic rings stabilize two peptide turns. Slow subsequent folding to a thermodynamically more stable two-turn a-helix drives the equilibrium to [{Pden}(2)-{(1,5)(4,8)-peptide}] (3), featuring two 22-membered rings. This transformation from unstructured peptide via turns to an a-helix suggests that metal clips might be useful probes for investigating peptide folding.

Discovery, structural determination, and putative processing of the precursor protein that produces the cyclic trypsin inhibitor sunflower trypsin inhibitor 1

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.

Structure of circulin B and implications for antimicrobial activity of the cyclotides

The solution structure of one of the first members of the cyclotide family of macrocyclic peptides to be discovered, circulin B has been determined and compared with that of circulin A and related cyclotides. Cyclotides are mini-proteins derived from plants that have the characteristic features of a head-to-tail cyclised peptide backbone and a knotted arrangement of their three disulfide bonds. First discovered because of their uterotonic or anti-HIV activity, they have also been reported to have activity against a range of Gram positive and Gram negative bacteria as well as fungi. The aim of the current study was to develop structure-activity relationships to rationalise this antimicrobial activity. Comparison of cyclotide structures and activities suggests that the presence and location of cationic residues may be a requirement for activity against Gram negative bacteria. Understanding the topological differences associated with the antimicrobial activity of the cyclotides is of significant interest and potentially may be harnessed for pharmaceutical applications.

Isolation and characterization of novel cyclotides from Viola hederaceae - Solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide

Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 ( vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional H-1 NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded β- sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and ∼ 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 μ m.

Isolation, solution structure, and insecticidal activity of Kalata B2, a circular protein with a twist: Do Mobius strips exist in nature?

A large number of macrocyclic miniproteins with diverse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using H-1 NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.

Discovery and characterization of a linear cyclotide from Viola odorata: Implications for the processing of circular proteins

Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule. (c) 2006 Elsevier Ltd. All rights reserved.

Modular alpha-helical mimetics with antiviral activity against respiratory syncitial virus

A 13-residue peptide sequence from a respiratory syncitial virus fusion protein was constrained in an alpha-helical conformation by fusing two back-to-back cyclic alpha-turn mimetics. The resulting peptide, Ac-(3 -> 7; 8 -> 12)-bicyclo-FP[KDEFD][KSIRD]V-NH2, was highly alpha-helical in water by CD and NMR spectroscopy, correctly positioning crucial binding residues (F488, I491, V493) on one face of the helix and side chain-side chain linkers on a noninteracting face of the helix. This compound displayed potent activity in both a recombinant fusion assay and an RSV antiviral assay (IC50 = 36 nM) and demonstrates for the first time that back-to-back modular alpha-helix mimetics can produce functional antagonists of important protein-protein interactions.