Cyclic Sunflower Trypsin Inhibitor-1 Research Articles

Buried treasure: biosynthesis, structures and applications of cyclic peptides hidden in seed storage albumins.

Covering: 1999 up to the end of 2017The small cyclic peptide SunFlower Trypsin Inhibitor-1 (SFTI-1) from sunflower seeds is the prototypic member of a novel family of natural products. The biosynthesis of these peptides is intriguing as their gene-encoded peptide backbone emerges from a precursor protein that also contains a seed storage albumin. The peptide sequence is cleaved out from the precursor and cyclised by the albumin-maturing enzymatic machinery. Three-dimensional solution NMR structures of a number of these peptides, and of the intact precursor protein preproalbumin with SFTI-1, have now been elucidated. Furthermore, the evolution of the family has been described and a detailed understanding of the biosynthetic steps, which are necessary to produce cyclic SFTI-1, is emerging. Macrocyclisation provides peptide stability and thus represents a key strategy in peptide drug development. Consequently the constrained structure of SFTI-1 has been explored as a template for protein engineering, for tuning selectivity towards clinically relevant proteases and for grafting in sequences with completely novel functions. Here we review the discovery of the SFTI-1 peptide family, their evolution, biosynthetic origin, and structural features, as well as highlight the potential applications of this unique class of natural products.

Sortase A-mediated synthesis of ligand-grafted cyclized peptides for modulating a model protein-protein interaction.

Specific ligand-grafted cyclic peptides are promising drug candidates that can modulate protein-protein interactions (PPIs) with increased proteolytic stability. In this study, we aimed to demonstrate that Sortase A (SrtA)-mediated peptide transpeptidation can be applied to produce bioactive sequence-grafted, stable, cyclic peptides. A naturally occurring cyclic peptide, sunflower trypsin inhibitor 1 (SFTI-1), was selected as the scaffold, and a tetrapeptide motif, Glu-Ser-Asp-Val (ESDV), was grafted into the scaffold as a model ligand. The linear precursor of the grafted peptide with SrtA-recognition motifs at the N- and C-termini was cyclized in good yield simply by co-incubation with SrtA. The ESDV-grafted cyclic SFTI-1 obtained was confirmed to have high stability against proteolysis by human serum and bound to the target PDZ2 domain of postsynaptic density-95 protein. An optimized sequence-grafted cyclic SFTI-1 could competitively suppress the interaction of PDZ2 with its natural ligand, the C-terminal peptide of the NR2B subunit of the N-methyl-D-aspartate receptor. These results show that a strategy combining peptide grafting into the SFTI-1 scaffold with SrtA-catalyzed cyclization can be a simple and effective method for producing stable peptide drugs.

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.

Enzymatic Cyclization of a Potent Bowman-Birk Protease Inhibitor, Sunflower Trypsin Inhibitor-1, and Solution Structure of an Acyclic Precursor Peptide

The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI-1[6,5] is approximately 9:1 regardless of whether trypsin is incubated with SFTI-1[6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[6,5] revealed high heterogeneity, and multiple species of SFTI-1[6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an iso-aspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.