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.
Marx
Ute C
UC
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.
Korsinczky
Michael L J
ML
Schirra
Horst Joachim
HJ
Jones
Alun
A
Condie
Barrie
B
Otvos
Laszlo
L
Craik
David J
DJ
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002417:Cattle; D006368:Helianthus / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D010456:Peptides, Cyclic / Q000737:chemistry*; D011487:Protein Conformation; D012639:Seeds / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D000596:Amino Acids; D010455:Peptides; D010456:Peptides, Cyclic; C448501:SFTI-1 peptide, sunflower; D014357:Trypsin