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The novelty of gene-expressed backbone cyclised proteins makes them an intriguing field of enquiry. Apart from their biological interest the application of backbone cyclisation in drug design and the utilisation of naturally occurring cyclic peptides as scaffolds also makes research in the area important for the development of proteins and peptides as therapeutic agents. Although a number of backbone cyclised proteins have now been identified, the manner in which they are produced, their evolutionary relationship and their relationship to their linear cousins for the most part remains unknown. However, a few key trends have emerged when the group is examined as a whole.
Backbone cyclised proteins and peptides adopt a number of folds and backbone cyclisation can lead to stabilisation of a protein. Apart from the resistance to exopeptidases that is conferred by removal of the N and C termini, many of the proteins examined in this chapter appear to be thermo-stabilised to some degree by backbone cyclisation. However from a drug design and bioavailability perspective it is important that protein drugs be at least partially resistant to a range of endoproteases. Although AS-48 exhibits thermostablity it, along with the sex pilus, are still vulnerable to several common endoproteinases, including trypsin amongst others. On the other hand SFTI-1 and the cyclotides are resistant to a wide range of adverse conditions. This suggests that the extreme stability is not only a factor of backbone cyclisation but also the rigidity of the cyclic fold. The high thermostability of the linear knottins, as evidenced by EETI, would appear to bear out this conjecture and it would be of great interest for a determination of what degree of resistance to endoproteases is possessed by the cyclic squash TIs. From a drug design perspective therefore while backbone cyclisation does appear to bring about stabilisation, resistance to a wide range of proteases appears to be dependent on the rigidity of the fold -- this can be enhanced by backbone cyclisation but it is not the only factor as evidenced by the flexibility of RTD-1. This has repercussions when considering the size and conformation of novel motifs utilising cyclic scaffolds.
Cyclic proteins and peptides are now found in many different organisms and an evolutionary line connecting them all seems implausible. At this stage, therefore, cyclisation of the protein backbone appears to be an adaptation that has risen independently on a number of different occasions. Evidence from RTD and the cyclic squash trypsin inhibitors suggests that fortuitous changes in precursors of linear proteins can bring about cyclisation without the need for a concomitant evolution of specialised processing machinery. Cyclic squash TIs appear to have arisen in only one species and appear to incorporate parts of the precursor, present in linear trypsin inhibitors, in the mature peptide. If no other cyclic squash trypsin inhibitors exist then assumedly minor mutations in this one species have brought about backbone cyclisation. In RTD-1 the insertion of a stop codon in an -defensin led to the advent of a backbone cyclised peptide, although in this case the added complexity of two independent genes being joined suggests that there is some added complexity in the processing mechanism. Adding further weight to this conjecture is the sex pilin VirB2 which is capable of being cyclised without any other plasmid borne protein, suggesting once again that endogenous enzymes are capable of mediating cyclisation in certain proteins. This strongly suggests that the important elements of cyclisation are the linear precursors of cyclic peptides and it is the interaction of these proteins with normal processing enzymes, such as proteases, that brings about backbone cyclisation.
SFTI-1 and the bacterial sex pilin system show the clearest evidence for proteases acting as mediators of backbone cyclisation. Cyclisation of linear isomers of SFTI-1 show that proteases are capable of mediating peptide bond formation. In the case of SFTI-1 the rigid structure of the peptide appears to prevent conformational change upon hydrolysis and hence drives the equilibrium of cyclised to linear in the direction of cyclised peptide. This rigidity most likely acts to keep the N and C termini in close proximity, enabling the reversal of the reaction. In the sex pilin the positioning of the TrbC intermediate in the plasma membrane ensures the proximity of the N and C termini so that cleavage of the tetrapeptide and consequent aminolysis by the amino terminal is possible. In the knottins, the two families that, so far, possess cyclised examples both have a Cys arrangement that would promote the proximity of the N and C termini during processing. Accordingly, two factors appear to be important for cyclisation -- the proximity of the N and C termini, as brought about by rigidity of the precursor structure or positioning in a plasma membrane, and the presence of residues in the linear precursor capable of interacting with a particular protease, either in a recognition role or as co-factors in the peptide bond forming reaction. To determine the validity of such a hypothesis the precursors of a range of closely related linear and cyclic peptides is required so that comparison of the sequential and structural characteristics can lead to an identification of the residues important for cyclisation.
An interesting test of this theory is to consider the case of the cyclotides. As discussed already, they are not widely spread within the plant kingdom, but are found in two distantly related plant families. The possibility exists that cyclotides are more common within the plant kingdom but have remained undetected or that linear homologues exist within the plant kingdom which are also not detected by current screening methods. If an ancestral gene is posited to explain the presence of the cyclotides in two disparate plant families then finding linear examples of cyclotides in other plant families would enable the elements important for cyclisation to be identified. Cyclisation arising in two different plant lineages such as the Rubiaceae and the Violaceae could demonstrate the relative simplicity of the cyclising reaction given the right conditions and would also explain why the distribution of cyclotides in the plant kingdom is so unusual.
Next: References Up: Introduction Previous: -defensins Jason Mulvenna