Two-step self-assembly of a spider silk molecular clamp
Molecular details that underlie mechanical properties of spider silk are of great interest to material scientists. We identified a three-state mechanism of self-association and an expanded structure of a spider silk protein that may contribute to elasticity of the fiber.
Silk fibers of web spiders are amongst nature’s toughest materials. Based on their low weight they supersede man-made high-tech material like Carbon or Kevlar. Material scientists are trying to reproduce the fiber in the laboratory, but with limited success1. The secrets of spider silk may be disclosed if forces and mechanisms that furnish the unprecedented strength and extensibility of the fiber are identified.
Spider silk is a result of hundreds of millions of years of evolution. Its synthesis is highly conserved across glands and species. The animal transforms soluble proteins, so-called spidroins, into solid silk during their passage through a tapering spinning duct. In the spinning duct, spidroins experience mechanical and chemical stimuli that induce phase and structural transitions. While the bulk of a spidroin is an unfolded polypeptide in solution, the terminal domains are regularly folded.
During past years our laboratory studied folding and association of spidroin N-terminal domains (NTDs). Our focus has been on the NTD from a nursery web spider, for which an atomic-detailed crystal structure was reported for the first time2. The C-terminal domain (CTD) has been neglected by us for a while. We embarked on the CTD because it shares many structural features with the NTD, but also shows some very interesting differences. Both NTD and CTD are five-helix bundles that have unusual amino acid composition. While the NTD exhibits a rather conventional association interface, the CTD forms intertwined chains and contains a covalent disulfide that connects domains permanently3. The fold resembles that of a knotted protein.
We started laboratory synthesis and mutagenesis of the CTD and soon realized that the domain behaved very differently compared to the NTD. While the NTD over-expressed in E. coli bacterial cells at high yield, the CTD required a fusion protein to boost synthesis. Our fluorescence reporter design and functional studies rely on mutagenesis. While the NTD was quite tolerant to the exchange of individual amino acids, the CTD did not at all like these mutations. It seemed that nature evolved a highly optimized sequence where every amino acid side chain was in place for a specific purpose. The CTD just didn’t want to be modified. After several months of trials we finally managed to identify sequence positions that tolerated modification. Our project gained momentum and spectroscopy commenced. The CTD reimbursed us with exceptionally clean data, which revealed an unusual mechanism of self-assembly.
We found that the molecular clamp geometry of intertwined helices connects spidroins in a very stable fashion. But the clamp was also a scaffold for the parking of peripheral helices, which were labile and unfolded easily. Knowing that elasticity of spider silk is associated with unfolding of helix, our results suggested that the CTD contributes to the characteristic elasticity of spider silk.
Our full story can be found in Nature Communications
1 Rising, A. & Johansson, J. Toward spinning artificial spider silk. Nature Chemical Biology 11, 309-315, (2015).
2 Askarieh, G. et al. Self-assembly of spider silk proteins is controlled by a pH-sensitive relay. Nature 465, 236-238, (2010).
3 Hagn, F. et al. A conserved spider silk domain acts as a molecular switch that controls fibre assembly. Nature 465, 239-242, (2010).