Abstract
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.
Highlights
Over recent years there has been much interest in the discovery of circular proteins in higher organisms (1) and in the development in synthetic approaches to cyclize proteins (2)
We show here that an acyclic permutant of sunflower trypsin inhibitor-1 (SFTI-1) open at its scissile bond, SFTI-1[6,5], functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine -trypsin
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
Summary
Over recent years there has been much interest in the discovery of circular proteins in higher organisms (1) and in the development in synthetic approaches to cyclize proteins (2). Unfavorable entropic losses upon binding to target proteins are significantly reduced, resulting in a thermodynamically more efficient binding interaction (1) These biological advantages of backbone cyclized peptides may lead to their use as scaffolds for the design of stable pharmaceuticals and pesticides (4). In vitro cyclization procedures are being developed for the synthetic production of circular proteins, little is known about the mechanisms and driving force behind in vivo cyclization of naturally occurring cyclic proteins. This is in part because limited knowledge about the locality of the NH2- and COOH termini of putative linear precursors of cyclic proteins makes predictions about cyclization mechanisms difficult. Zablotna et al (14) recently reported that there is essentially no difference between the inhibitory poten-
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