Evolutionary origins of a bioactive peptide buried within Preproalbumin

Abstract

The de novo evolution of proteins is now considered a frequented route for biological innovation, but the genetic and biochemical processes that lead to each newly created protein are often poorly documented. The common sunflower (Helianthus annuus) contains the unusual gene PawS1 (Preproalbumin with SFTI-1) that encodes a precursor for seed storage albumin; however, in a region usually discarded during albumin maturation, its sequence is matured into SFTI-1, a protease-inhibiting cyclic peptide with a motif homologous to unrelated inhibitors from legumes, cereals, and frogs. To understand how PawS1 acquired this additional peptide with novel biochemical functionality, we cloned PawS1 genes and showed that this dual destiny is over 18 million years old. This new family of mostly backbone-cyclic peptides is structurally diverse, but the protease-inhibitory motif was restricted to peptides from sunflower and close relatives from its subtribe. We describe a widely distributed, potential evolutionary intermediate PawS-Like1 (PawL1), which is matured into storage albumin, but makes no stable peptide despite possessing residues essential for processing and cyclization from within PawS1. Using sequences we cloned, we retrodict the likely stepwise creation of PawS1's additional destiny within a simple albumin precursor. We propose that relaxed selection enabled SFTI-1 to evolve its inhibitor function by converging upon a successful sequence and structure.

Figure 1.

Structural Similarity between SFTI-1 and the Inhibitory Arm of Bowman-Birk Inhibitors (A) Sequence of the backbone cyclic and disulfide bonded SFTI-1 with the line joining Cys residues denoting a disulfide bond and the line joining Gly and Asp denoting the cyclic backbone. Circled numbers from SFTI-1 (Cys-3, Lys-5, Pro-8, Pro-9, and Cys-11) are aligned with the BBI WebLogo (B) and used to mark their locations within the structural overlay (C). (B) WebLogo summary of 150 aligned short segments of BBIs. (C) SFTI-1 (stick format) overlaid upon the inhibitory loops of 10 BBIs (line format). (D) Angiosperm phylogeny based on rbcL sequences and adapted from Mylne et al. (2012). The families containing BBIs are shown (Fabaceae or legumes, and Poaceae or grasses). The phylogenetically separate position of the BBI-loop mimic sunflower SFTI-1 is also shown.

Figure 2.

PawS1 Proteins from the Daisy Family (Asteraceae) and Their Buried Peptides (A) PawS1 domains include the ER signal, PDP region, and the small (SSU) and large subunits (LSU) of mature albumin based on sunflower PawS1 (Mylne et al., 2011). (B) Percentage (0 to 100%) identity graph for 28 PawS1 protein sequences showing weaker conservation around the PDP region (dashed box). The arrowhead beneath the graph indicates the position where four PawS1 sequences have 100 amino acid internal expansions. See Supplemental Figure 5 for an alignment of full-length sequences. (C) Alignment of selected in vivo confirmed (closed circles) and gene-predicted (open circles) PDPs showing little conservation except the N-terminal Gly, C-terminal Asp, a Cys-pair, and central Pro-Pro. The BBI-loop mimics are marked with asterisks. (D) Phylogenetic tree (Supplemental Data Sets 3 and 4) including species used in this study and showing PDPs are common to the Heliantheae and Millerieae tribes estimated to have diverged (dashed circle) 18 million years ago (Ma) (Supplemental Figure 6). Tribe names are black, whereas subtribe names are in red/gray. [See online article for color version of this figure.]

Figure 3.

Structures of PDPs and in Vivo Peptide-Processing Constraints for SFTI-1. (A) Ribbon representation of NMR-derived structures for novel PDPs alongside sunflower SFTI-1 (Korsinczky et al., 2001) and SFT-L1 (Mylne et al., 2011). (B) In vivo mutagenesis data from previous work (Mylne et al., 2011) and this study (asterisked) reveals which changes are tolerated biologically (green), perturb peptide processing (black), or specifically perturb the cyclization reaction (red). (C) PawL1 from A. montana shares many of the critical residues for peptide maturation but lacks Cys residues and does not make a stable peptide.

Figure 4.

The Am-PawL1 Gene Is Transcribed, Translated, and Processed into Four Subtly Different Heterodimeric Albumins. (A) RT-PCR confirmation of Am-PawL1 expression. (B) Gel image of fast protein liquid chromatography separation of an albumin-rich extract from A. montana seeds. Two fast protein liquid chromatography fractions (asterisks) were further separated by HPLC, and two fractions at 41 min (asterisks) were identified by endo-GluC or trypsin digestion and MS/MS to contain Am-PawL1 mature albumin. For all MS/MS, see Supplemental Tables 10 and 11 for a complete list of ions. (C) Endo-GluC and tryptic peptide fragments (underlined) mapped onto Am-PawL1. The predicted Am-PawL1 ER signal is highlighted in pink, the mature albumin SSU in green, and the mature albumin LSU in orange. (D) Am-PawL1 SSU and LSU sequences. (E) Expected masses for the four SSU/LSU combinations and the abbreviations for each. (F) ESI-TOF-MS spectrum of the fractions containing native Am-PawL1 albumin. Ions corresponding to the expected masses for each conformation are indicated. (G) ESI-TOF-MS spectrum of the reduced and alkylated fraction containing the Am-PawL1 albumin. (H) ESI-TOF-MS/MS spectrum of the peak corresponding to SSU1 (820.9 D). 1+ (purple), 2+ (green), and 3+ (orange) b and y ions are indicated. (I) ESI-TOF-MS/MS spectrum of the peak corresponding to SSU2 (835.1 D).

Figure 5.

Model for PawS1 Evolution and the Evolutionary Recurrence of the BBI Inhibitory Motif. (A) A model proposed for the progressive evolution of the BBI mimicking peptide SFTI-1 from within a standard preproalbumin (PAL) with the key sequence changes shown on the right. (B) Precursor proteins highlighting the location of the inhibitory loop within the mature BBI (XP_003533609), SFTI-1 and its adjacently matured napin, and the mature ORB peptide from amphibian frog skin (DQ672940). ER denotes the ER signal sequence. (C) Sequences of SFTI-1, ORB, and four partial BBIs. (D) Structural overlay of SFTI-1 (1SFI), frog ORB lacking two N-terminal and one C-terminal residues (2O9Q), and a BBI from soybean (1BBI).