Two Proteins for the Price of One: Structural Studies of the Dual Destiny Preproalbumin with Sunflower Trypsin Inhibitor-1

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 sunflower contain a sequence that is released as a macrocyclic peptide during post-translational processing. The mechanism by which such peptides emerge from linear precursors 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 PreproAlbumin With Sunflower trypsin inhibitor-1 (PawS1), and provide new insights into the processing of this remarkable dual-destiny protein. In seeds PawS1 is matured by asparaginyl endopeptidases (AEP) into the cyclic peptide Sunflower Trypsin Inhibitor-1 (SFTI-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 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.