Abstract
Seed storage proteins are both an important source of nutrition for humans and essential for seedling establishment. Interestingly, unusual napin-type 2S seed storage albumin precursors in sunflowers contain a sequence that is released as a macrocyclic peptide during post-translational processing. The mechanism by which such peptides emerge from linear precursor proteins has received increased attention; however, the structural characterization of intact precursor proteins has been limited. Here, we report the 3D NMR structure of the Helianthus annuus PawS1 (preproalbumin with sunflower trypsin inhibitor-1) and provide new insights into the processing of this remarkable dual-destiny protein. In seeds, PawS1 is matured by asparaginyl endopeptidases (AEPs) into the cyclic peptide SFTI-1 (sunflower trypsin inhibitor-1) and a heterodimeric 2S albumin. The structure of PawS1 revealed that SFTI-1 and the albumin are independently folded into well-defined domains separated by a flexible linker. PawS1 was cleaved in vitro with recombinant sunflower HaAEP1 and in situ using a sunflower seed extract in a way that resembled the expected in vivo cleavages. Recombinant HaAEP1 cleaved PawS1 at multiple positions, and in situ, its flexible linker was removed, yielding fully mature heterodimeric albumin. Liberation and cyclization of SFTI-1, however, was inefficient, suggesting that specific seed conditions or components may be required for in vivo biosynthesis of SFTI-1. In summary, this study has revealed the 3D structure of a macrocyclic precursor protein and provided important mechanistic insights into the maturation of sunflower proalbumins into an albumin and a macrocyclic peptide.
Highlights
Edited by Joseph JezSeed storage proteins are both an important source of nutrition for humans and essential for seedling establishment
Of the four helical segments in PawS1, helix I was located in the small subunit (SSU), whereas the three remaining helical segments were all located in the large subunit (LSU)
The cysteine connectivity is conserved among seed storage albumins and features two interchain disulfide bonds connecting the small and the large subunit (I–V and II–III) and two intrachain disulfide bonds stabilizing the large subunit (IV–VII and VI–VIII)
Summary
Seed storage proteins are both an important source of nutrition for humans and essential for seedling establishment. Unusual napin-type 2S seed storage albumin precursors in sunflowers contain a sequence that is released as a macrocyclic peptide during post-translational processing. The prototypic seed albumin is Brassica napus (canola) napin, which begins as a 178-residue protein that exits the endoplasmic reticulum as a 157-amino acid protein It is further processed through the removal of two joining peptides and the C-terminal residue by the action of endoproteases called asparaginyl endopeptidases (AEPs), yielding the “mature” napin. Preproalbumin with SFTI-1 (sunflower trypsin inhibitor-1) named PawS1 and a closely related PawS2 are matured into typical heterodimeric albumins with one small and one large subunit and yield the 14-residue SFTI-1 (Fig. 1) and the 12-residue SFT-L1, respectively [10] Both peptides are head-to-tail peptide macrocycles with potential applications in drug design [11]. Despite the interest in these systems, the significance of the structural context in which the cyclic peptide sequence is embedded is poorly understood, and the structural characterization of intact precursor proteins for cyclic peptide families has been limited
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