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

Abstract

The most potent known naturally occurring BowmanBirk 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 ␤-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI-1[6,5] is ϳ9:1 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 ␤-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an isoaspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.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). In general, backbone cyclic peptides have several advantages over their non-cyclic counterparts. They are resistant to attack by exopeptidases, making them less vulnerable to degradation and can have an increased thermal stability (3). Also, 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).The new generation of circular peptides/proteins discovered in the last few years differs from previously known cyclic peptides such as cyclosporins in that the latter are generally not direct gene products but are synthesized in bacteria by multifunctional enzymes and often contain non-conventional amino acids (5). By contrast, recently discovered circular miniproteins such as the plant cyclotides (6) are gene products that are post-translationally processed to cyclize their conventional peptide backbone (7). Although in vitro cyclization procedures are now being developed for the synthetic production of circular proteins, little is known about the mechanisms and driving force behind in vivo cyclization of naturally o...

Selective Inhibition of the Lectin Pathway of Complement with Phage Display Selected Peptides against Mannose-Binding Lectin-Associated Serine Protease (MASP)-1 and -2: Significant Contribution of MASP-1 to Lectin Pathway Activation

“…The crystal structure of the free MASP-2 enzyme (40) showed that in the absence of bound substrate or inhibitor, the substrate binding groove on the enzyme is largely obstructed by long surface loops. Lacking structural information about the positions of these loops in a complex, we decided to design the library based on the smallest known natural trypsin inhibitor scaffold of the SFTI (27)(28)(29)(30).…”

“…Our target proteases were the MASP-1 and MASP-2, the initiator proteases of the lectin pathway. As a starting point for inhibitor selection, we used the sunflower trypsin inhibitor (SFTI), which has the shortest scaffold (only 14 aa) among the known natural SP inhibitors (27)(28)(29)(30). The selected peptides inhibit exclusively the lectin pathway among the three activation pathways of complement.…”

Biological Synthesis of Circular Polypeptides

“…These include the use of protein splicing catalyzed by inteins (16) or proteases (41,42) as well as thioester-based ligation using either modified inteins (27,29,30) or genetic code reprogramming (46). The ability to produce circular polypeptides using the tools of molecular biology has multiple biotechnological applications (Scheme 1).…”

“…MCoTI cyclotides and SFTI-1 are potent trypsin inhibitors, with K i values in the picomolar and nanomolar range, respectively. In both cases, the proteolytic enzyme trypsin was able to resynthesize the peptide bond between the P1 and P1Ј residues to form the native circular polypeptides (41,42). Although this approach has not yet been tested in vivo, there is mounting evidence that protease-catalyzed protein splicing is used in the biosynthesis of these naturally occurring circular polypeptides in plants.…”

Prospects for the design of new therapeutically significant protease inhibitors based on knottins and sunflower seed trypsin inhibitor (SFTI 1)

“…Ингибиторная петля, Thr4-Ile10, содержит сайт возможного расщепления трипсином Lys5-Ser6. Однако бoльшая часть молекул ингибитора остаётся негидролизованной: отношение количества нативного ингибитора к количеству гидролизованного ингибитора составляет 9:1 [65]. Вторичная петля, пентапептидный фрагмент, не участвует прямо во взаимодействии с протеазой, но стабилизирует структуру всего пептида за счёт формирования водородных связей с остатками ингибиторной петли.…”