Two proteins for the price of one: Structural studies of the dual-destiny protein preproalbumin with sunflower trypsin inhibitor-1

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 <i>Helianthus annuus</i> PawS1 (<u>p</u>repro<u>a</u>lbumin <u>w</u>ith <u>s</u>unflower trypsin inhibitor-<u>1</u>) 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 (<u>s</u>un<u>f</u>lower <u>t</u>rypsin <u>i</u>nhibitor-<u>1</u>) 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 <i>in vitro</i> with recombinant sunflower HaAEP1 and <i>in situ</i> using a sunflower seed extract in a way that resembled the expected <i>in vivo</i> cleavages. Recombinant HaAEP1 cleaved PawS1 at multiple positions, and <i>in situ</i>, 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 <i>in vivo</i> 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.