Structural conversion of the spidroin C-terminal domain during assembly of spider silk fibers

Abstract

The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to β-sheet transition upon fiber formation and highlight the helix N^o4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix N^o4 has the ability of pH-induced conversion into β-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly.

Fig. 1. Overview of spidroin and CT domain (E. Australis) structure.

a Schematic illustration of full-length major ampullate spidroins, including: N-terminal domain (NT), repetitive central region (poly-Ala/Gly), and C-terminal domain (CT). b The comparative modeling (homology model) of CT domain of E. australis MaSp1, shown with chain A in blue, chain B in orange and the inter-chain disulfide bridge represented in yellow. The amino acid residues forming salt bridges are visualized with stick representation where the negative charges are red and the positive charges are blue. c CD spectroscopy of CT domain (MaSp1 E. australis) at pH 8 presents the canonical α-helix pattern with minima at 222 nm and 208 nm. d The sequence of CT with the α-helices indicated, and the conservation score according to ConSurf . The images were rendered using Pymol.

Fig. 2. FTIR spectra of CT fiber in the amide I region.

a Fibers of the recombinantly produced CT domain obtained by a stress-relaxation cycle method. b Second derivative of the absorbance spectrum (black) and the fit (red) for the CT fiber. c Absorbance spectrum of the CT fiber (black), the fit (red) with the same fit model as in (b), and the fitted component bands (dotted lines). Other secondary structures refers to irregular, turns, bends, and other helix types.

Fig. 3. The X-ray diffraction pattern of recombinant silk protein fibers.

Individual diffraction patterns (a, b) and azimuthally averaged pattern (c, d) of fibers from the CT domain (a, c) and the whole 4RepCT construct (b, d). The size of the side chains determines the packing of crystalline regions and thereby the inter-sheet distances.

Fig. 4. 2D DARR spectra (50 ms mixing time) of CT fiber.

The secondary structure components of the amino acid residues of CT in fiber form based on the chemical shifts are indicated by the colors: β-sheet in red, α-helix in blue, and random coil in black.

Fig. 5. Predictions of fibrillation-prone regions of the CT sequence.

The CT sequence with the positions of the helices in the model of the soluble CT shown as comparison. Amino acid residues with β-sheet chemical shifts from ssNMR are indicated in red. Computational analysis using ZipperDB fibrillation propensity and theoretical inter-sheet distances in combination with TANGO cross-β aggregation at pH 7 and pH 4 suggest hotspots for possible formation of β-sheets.

Fig. 6. The secondary structure evaluation of CT domain helix N^o4 suggests a structural shift of the peptide to β-sheet.

a CD spectra of the helix N^o4 peptide at different pH values. b Representative AFM image of nanofibrils formed by the helix N^o4 peptide when incubated at pH 3.1.

Fig. 7. The proposed model for α-helix to β-sheet structural conversion of the CT domain and the helix N^o4 peptide.

The complete CT domain forms fibers, as presented with scanning electron microscopy (left side). The C-terminal segment helix N^o4 refolds from α-helix to β-sheet to form a supramolecular β-sheet structure at a hydrophobic/hydrophilic interface. Helix N^o4 (peptide CT_51–80) has been shown to form fibrils under acidic pH, as shown by AFM (right side). Glu is shown in red, Arg in blue, Cys in orange, and helix N^o4 in yellow.