The cystine knot motif in toxins and implications for drug design
Introduction
The cystine knot is a fascinating structural motif in which an embedded ring formed by two disulfide bonds and their connecting backbone segments is threaded by a third disulfide bond, as illustrated in Fig. 1. The motif has been formally identified for less than a decade but is now known to occur in a wide variety of peptides and proteins and is relatively common in small cysteine-rich toxins. The cystine knot appears to engender a particularly high degree of stability to molecules that contain it and thus offers potential as a valuable framework in protein engineering applications. In this article we describe the structural features and biological activities of toxin molecules containing this motif, along with chemical methods for their synthesis. The ability to synthesize and determine structures of peptides containing the cystine knot enables a range of potential protein engineering and drug design applications. Examples of these are described.
Section snippets
Discovery of the cystine knot
The term “cystine knot’ was first introduced in 1993 (McDonald et al., 1993, Murray-Rust et al., 1993) in reviews describing recently determined structures for several growth factors, including nerve growth factor (NGF), transforming growth factor β2 (TGFβ2), and platelet derived growth factor BB (PDGF-BB). It was noted that these proteins contained an unusual motif comprising an embedded disulfide ring and a penetrating disulfide bond. The apparently knotted arrangement of disulfide bonds was
Toxin molecules containing the cystine knot
Table 1 lists a representative collection of toxin molecules that incorporate a cystine knot within their structures. These include peptides from plants, animals and fungi. The sequences are aligned based on the six conserved Cys residues that make up the knot (labeled I to VI using the same convention as in Fig. 2) and are ordered based on the size of the embedded ring within the knots. The loops referred to in the table header are the backbone segments between successive Cys residues. Note
Structures of cystine knot toxins
Because small disulfide rich peptides are difficult to crystallize, most of the information on structures of toxins containing the cystine knot has come from solution NMR studies. Fig. 4 shows the structures of two well-characterized examples, MVIIA and kalata B1, as representatives of the ICK and CCK families respectively. The figure shows that the predominant secondary structure element in either case is a distorted triple stranded β-sheet that is intimately associated with the cystine knot.
The cyclic cystine knot
We noted above that cystine knot peptides include some unusual molecules that contain a circular peptide backbone. In particular, it has now been shown that kalata B1 is just one member of a new family of macrocyclic peptides we have termed the cyclotides (Craik et al., 1999). These peptides are approximately 30 amino acids in length and are isolated from plants in the Rubiaceae and Violaceae families. They display an interesting range of biological activities which led to their initial
Topological considerations — unknotting of the cystine knot
There is a fundamental topological difference between the cyclic and acyclic cystine knot proteins. Although commonly referred to as knots, the latter are in fact not knotted in a mathematical sense (Mansfield, 1994, Mislow and Liang, 1996) and are topologically simple (Mao, 1993). This means that the conventional disulfide connectivity shown in Fig. 8A may be drawn in two dimensions on a non-crossing diagram, such as in Fig. 8B. By contrast, the CCK peptides are topologically complex, and
Permutation of cystine knot structures
As the cystine knot occurs as an embedded motif in a wide variety of cyclic and non-cyclic peptide toxins, it is of interest to examine the role of permutation of the protein termini in acyclic derivatives, or equivalently, of acyclic permutation in the cyclic derivatives. The basic question is do the positions of the peptide termini relative to cystine knot motif matter? To answer this we recently synthesized a series of truncated acyclic permutants of kalata B1 in which each of the six
ICK peptides
Cystine knot peptides represent an attractive proposition for use as scaffolds in drug design applications because of the innate stability of the fold and the extreme variability of sequences that can be accommodated. However, to effectively exploit cystine knot peptides in such applications an understanding of their synthesis and folding is required.
The synthesis and folding of conotoxins have been extensively studied because of their potential for therapeutic applications. It was originally
Drug design and molecular engineering
The exceptional stability and well-defined scaffolds of cystine knot peptides mean that they have promising pharmaceutical applications, both as lead molecules themselves, or as molecular frameworks for pharmaceutical design. Here we outline some applications of these peptides in pain therapy, as antibacterials and as potential antiviral agents, and finish with some molecular engineering applications.
The use of cystine knot peptides in pain therapy relates to the ability of MVIIA and related
Acknowledgments
This work was supported in part by a grant from the Australian Research Council (DJC). DJC is an Australian Research Council Senior Fellow. The Institute for Molecular Bioscience is a Special Research Centre of the ARC.
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