-defensins next up previous contents
Next: Summary and Common Themes Up: Other Cyclic Peptides Previous: Sex pilus subunits

$ \theta $-defensins

The defensins are a class of small Cys rich protein widely distributed throughout plant, animal, insect, bird and mammal species including humans [160]. It is believed that this class of peptide is involved in innate immunity, conferring resistance to pathogens via antimicrobial and anti-viral activity [161]. Two families of, non-cyclic, defensins have been discovered in mammals -- the $ \alpha$ and $ \beta$ defensins. These peptides range in size from 29 to 42 amino acids and possess a three disulphide motif with different connectivity in each class. Despite the different connectivity of the disulphide bonds both class of defensin displays a similar overall fold [162]. Recently a third class of neutrophil expressed defensin was discovered in Macaque monkeys. This class of defensin, termed rhesus $ \theta $-defensin-1 (RTD-1), is distinguished by possessing a high content of Arg and Cys residues and, surprisingly, head-to-tail cyclisation of its peptide backbone [163]. The 18 residue molecule is stabilised by three disulphide bonds between its six Cys residues, however, unlike the cyclotides, the disulphide bonds are arranged in a laddered topology.

Figure 1.21: Sequence and Structure of RTD-1 A. Structure of RTD-1 (PDB code 1HVZ) showing the laddered arrangement of disulphide bonds. This arrangement of disulphides allows RTD-1 flexibility around its central region as seen in B. C. shows an alignment of RTD-1a and RTD-1b with the $ \alpha$-defensin HNP-4 and the $ \alpha$-defensin-like pseudogene Humtd$ \psi$1. Each RTD gene is a truncated version of the myeloid $ \alpha$-defensin and shares 88% sequence identity with the human pseudogene. Disulphide connectivities for RTD-1 and HNP-4 have been indicated.

The solution structure of RTD-1 has been solved [164] and, in contrast to the rigid, well defined structures of SFTI-1 and the cyclotides, the structure of RTD-1 appears to be somewhat flexible, displaying the ability to bend about its central region (Figure 1.21). It is interesting to contrast RTD-1 with SFTI-1, which is braced by a single disulphide. In SFTI-1 the rigid structure shows no lateral flexibility, probably as a consequence of its hydrogen bonding network. Studies of the anti-microbial activity of RTD-1 show that the peptide can bind to cell membranes in two different orientations [165]. In only one of these orientations was weak membrane thinning observed and it has been speculated that the flexibility of RTD-1 allows it to bind to polar headgroups without the disruption caused by the more rigid $ \alpha$ and $ \beta$ defensins [166]. Consistent with this RTD-1 has been found to possess very low cytotoxicity for human blood mononuclear cells [167]. Hence the flexibility of RTD-1 may reflect its physiological role, just as the rigid structure of SFTI-1 reflects its role as a trypsin inhibitor. The flexibility of RTD also highlights the importance of factors other than disulfides and backbone cyclisation in conferring conformational rigidity.

The most fascinating aspect of the $ \theta $-defensins is their biosynthesis. RT-PCR experiments designed to isolate the gene for RTD-1 produced two genes, RTD-1a and RTD-1b, each of which encoded a nine residue peptide that comprised half of the mature RTD-1 [163]. These genes encode a precursor with a signal sequence, a pro-peptide, a nine residue mature segment that contains three of the six Cys residues that comprise the mature peptide and a three residue tail. Each gene is a homologue of a myeloid $ \alpha$-defensin, differing only in the presence of a premature stop codon 3 residues C-terminal to the final Cys residue (Figure 1.21) and each gene possesses the typical 3-exon/2-intron structure of all known myeloid defensin. Furthermore, the nucleotide sequence of each gene is 88% identical to that of a human $ \alpha$-defensin-like pseudogene. It is fascinating, therefore, to contemplate that RTD-1 is derived from two mutated halves of $ \alpha$-defensin genes.

Figure 1.22: Possible biosynthetic route of RTD1 The two gene structure of RTD-1 implies that two cleavage reactions for each precursor must take place to remove the N and C-terminal pro-peptides prior to ligation of the free termini. How the two separate halves of RTD-1 are organised prior to the cyclisation reaction is not known although disulphide formation prior to the cleavages/ligations could be one possible method of ensuring the proximity of the ligated termini.

The biosynthetic pathway implied by this genetic structure is set out in Figure 1.22. After signal sequence cleavage the nine residue domain is cleaved both C-terminally and N-terminally to release the tail and pro-peptide respectively. Either concomitantly or subsequently to these cleavage reactions two ligation reactions are required to tie the ends of the two peptides together to yield the mature RTD-1. How the two different precursors are organised and oriented to produce mature RTD-1 is not known although the formation of disulphide bonds prior to the ligations would appear to be a logical step. The biosynthetic pathway also allows for the production of two other species of homodimeric conformations and both of these peptides, RTD-2(1b/1b) and RTD-3 (1a/1a) have been identified in rhesus monkey bone marrow [168] and peripheral blood leukocytes in which they appear to be expressed in a 30:1:2 ratio of RTD 1-3 [169]. A third pro-RTD gene was predicted by cDNA cloning [167] and recent studies have provided evidence for expression of at least two of the possible three additional $ \theta $-defensins [166].

Like most of the other cyclising reactions examined in this chapter the precursor of RTD both a C-terminal and an N-terminal cleavage reaction is required prior to cyclisation and once again the role that each of these segments plays in the reaction is unknown. The requirement for cleavage implies the presence of a protease and as discussed earlier, proteases can also catalyse the ligation of peptide bonds. Indeed in the case of the pilin subunit the cleavage and ligation are thought to be dependent on each other. In RTD the entire reaction is further complicated by the necessity of positioning the two independent halves of the mature peptide prior to the cyclising reaction. It is interesting to note however that the mutation that gave rise to the circular RTD was a stop codon in a characterised gene. It seems unlikely that this mutation and machinery to take advantage of this mutation would arise simultaneously, so once again the processing of RTD is likely to be the result of enzymes that are part of the regular biosynthetic pathways of the organism containing it. This consideration also adds strength to the notion that changes in the precursor sequence of linear peptides leads to cyclised proteins and hence the determination of a range of precursors that lead to cyclic peptides is of paramount importance in unravelling the general principles of protein cyclisation.

next up previous contents
Next: Summary and Common Themes Up: Other Cyclic Peptides Previous: Sex pilus subunits
Jason Mulvenna