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 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.

Keywords: asparaginyl endopeptidase (AEP); cyclic peptide; nuclear magnetic resonance (NMR); plant biochemistry; post-translational modification (PTM); preproalbumin with SFTI-1 (PawS1); protein processing; seed storage albumin; sunflower trypsin inhibitor-1 (SFTI-1).

Figure 1.

Schematic of the domains of the preproalbumin PawS1 from H. annuus. The N-terminal signal peptide domain is highlighted in pink, the SFTI-1 peptide domain in cyan, the SSU of the albumin domain in green, and the LSU in orange. During processing, the SFTI-1 sequence is liberated and cyclized into a 14-residue peptide with one disulfide bond and a cyclic backbone. The cysteine connectivity highlighted by connecting lines (I–V, II–III, IV–VII, and VI–VIII within the albumin sequence) is conserved among plant 2S albumins.

Figure 2.

Expression and purification of PawS1. A, 1D gel showing His₆-TEV-PawS1 fusion protein before nickel-Sepharose HisTrap purification. Lanes are from the same gel but moved next to each other for clarity. B, RP-HPLC chromatogram showing purification of PawS1 after removal of N-terminal His₆ fusion tag by TEV protease. C, analytical RP-HPLC trace showing ∼95% pure protein judged by the single uniform peak. D, LC-MS of ¹³C- and ¹⁵N-labeled PawS1 purified by RP-HPLC.

Figure 3.

Sequence, 2D ¹H-¹⁵N HSQC spectrum acquired at 25 °C and 600 MHz, and secondary Hα chemical shifts for PawS1. A, sequence and numbering of the studied PawS1 protein showing the N-terminal SFTI-1 domain highlighted in cyan, the SSU in green, and the LSU in orange. Linker peptides shown in black connect SFTI-1 and the SSU as well as the SSU and the LSU of the heterodimeric albumin. B, peaks are labeled with the residue numbers of their corresponding backbone amides. Resonances originating from Asn/Gln and folded Arg side chains are highlighted by boxes. C, secondary shifts are observed chemical shifts subtracted by random coil chemical shifts and are indicative of secondary structure. The secondary Hα chemical shifts of the N-terminal SFTI-1 domain show the same trend as cyclic SFTI-1. Positive secondary shifts are indicative of β-sheet. Both the small and large subunits of the albumin domain show extensive stretches of negative secondary shifts, indicating a helical structure.

Figure 4.

3D solution NMR structure of PawS1. A, ensemble of the 20 structures representing PawS1 superimposed over the SFTI-1 domain. B, ensemble of the 20 structures representing PawS1 superimposed over the albumin domain. The SFTI-1 domain is shown in blue, the small subunit in green, the large subunit in orange, and the linker peptides GLDN and LRMAVEN in black. C and D, three structures from the ensemble are highlighted to clarify the range of conformations observed, with the remainder of the structures shown as transparent. Movie files highlighting the conformational rearrangements within the ensemble are provided as supplementary information.

Figure 5.

Heteronuclear ¹H-¹⁵N steady-state NOE data for ¹⁵N-labeled PawS1. *, proline residues Pro⁸, Pro⁹, Pro¹³, Pro¹⁹, Pro⁵⁰, and Pro¹⁰⁷ lack amide protons. #, the amide proton for Gln⁹⁸ was not identified. +, the peak for Ser⁶ was not detected in the NOE experiment, and thus a ratio could not be calculated.

Figure 6.

In vitro digest of PawS1 with sunflower HaAEP1. MALDI MS spectra showing cleavage products after incubation of PawS1 with HaAEP1 for 92 h at 37 °C. A, sunflower HaAEP1 cleaves the PawS1 after Asn¹⁸ and produces the linear SFTI-GLDN peptide. B, HaAEP1 also cleaves between the two subunits of the albumin PawS1 after Asn⁴⁹, which results in a mass shift of +18 Da. C, this cleavage can also be seen in full-length PawS1, resulting in a mass shift of +18 Da. Observed (M_o) and calculated (M_c) monoisotopic masses (Da; [M + H]⁺) are listed for SFTI-GLDN, and observed and calculated average masses (Da; [M + H]⁺) are listed for the albumin PawS1 and PawS1, respectively. The red line represents MALDI MS data of PawS1 incubated with HaAEP1 in AEP activity buffer. The blue line represents the control, MALDI MS data of PawS1 incubated without HaAEP1 in AEP activity buffer.

Figure 7.

In situ digest of PawS1 with sunflower seed extract. A, MALDI MS spectra showing full-length PawS1 during incubation with sunflower seed extract at 37 °C. After 2 h, cleavage had occurred after the linker peptide LRMAVEN, between the two subunits of PawS1. After 5 h, most of the PawS1 linker region was cleaved. B, after 5 h, fully matured PawS1 heterodimeric albumin with SFTI-GLDN and the LRMAVEN linker removed was also observed. The red line represents MALDI MS data of PawS1 incubated with sunflower seed extract in AEP activity buffer. The blue line represents the control, MALDI MS data of the sunflower seed extract incubated in AEP activity buffer for 5 h at 37 °C without PawS1 protein.

Figure 8.

2D ¹H-¹⁵N HSQC spectra of PawS1 incubated in in situ sunflower seed extract acquired at 37 °C and 600 MHz after 0 h, 7 h, and 10 days. Blue peaks represent ¹H-¹⁵N HSQC data of PawS1 incubated with in situ sunflower seed extract at 0 h. Red peaks represent ¹H-¹⁵N HSQC data of PawS1 incubated with in situ sunflower seed extract at 7 h. Green peaks represent ¹H-¹⁵N HSQC data of PawS1 incubated with in situ sunflower seed extract at 10 days. *, C-terminal residues of the intermediate and fully processed forms.

Figure 9.

Comparison of the NMR structure PawS1 with the sunflower albumin SESA3 and SFTI-1. A, the albumin domain of PawS1, showing SSU in green and LSU in orange, is superimposed on the sunflower albumin SESA3 shown in gray (PDB code of SESA3: 1S6D) (28). Helices are numbered with Roman numerals, and selected residues are labeled for clarity. B, the SFTI-1 domain of PawS1, shown in blue, is superimposed on the solution NMR structure of SFTI-1, shown in cyan (PDB code of SFTI-1: 1JBL) (18).