Structural Characterization of Minor Ampullate Spidroin Domains and Their Distinct Roles in Fibroin Solubility and Fiber Formation

Abstract

Spider silk is protein fibers with extraordinary mechanical properties. Up to now, it is still poorly understood how silk proteins are kept in a soluble form before spinning into fibers and how the protein molecules are aligned orderly to form fibers. Minor ampullate spidroin is one of the seven types of silk proteins, which consists of four types of domains: N-terminal domain, C-terminal domain (CTD), repetitive domain (RP) and linker domain (LK). Here we report the tertiary structure of CTD and secondary structures of RP and LK in aqueous solution, and their roles in protein stability, solubility and fiber formation. The stability and solubility of individual domains are dramatically different and can be explained by their distinct structures. For the tri-domain miniature fibroin, RP-LK-CTDMi, the three domains have no or weak interactions with one another at low protein concentrations (<1 mg/ml). The CTD in RP-LK-CTDMi is very stable and soluble, but it cannot stabilize the entire protein against chemical and thermal denaturation while it can keep the entire tri-domain in a highly water-soluble state. In the presence of shear force, protein aggregation is greatly accelerated and the aggregation rate is determined by the stability of folded domains and solubility of the disordered domains. Only the tri-domain RP-LK-CTDMi could form silk-like fibers, indicating that all three domains play distinct roles in fiber formation: LK as a nucleation site for assembly of protein molecules, RP for assistance of the assembly and CTD for regulating alignment of the assembled molecules.

Evidence from Multiple Species that Spider Silk Glue Component ASG2 is a Spidroin

“…The AgSp1 (ASG2) repetitive region is followed by a linker region that varies in length across species ( Fig. 5c ), as has been observed in some spidroin family members 33 34 . Unlike the linker regions, spidroin carboxyl-terminal regions are conserved in length and structure 34 35 36 .…”

Analysis of repetitive amino acid motifs reveals the essential features of spider dragline silk proteins

“…Consistent with MaSp sequences from other taxa [ 51 ], the different Tetragnatha MaSp-like subtypes all harbored the charged residues R52, D93 (or E93) and E101, as well as the disulfide-forming C92 residue within the CTD ( S1 Fig ). Variations among the CTDs included the lack of conserved R43 residue in subtypes A and E, which has been shown to participate in salt bridge formation with an acidic residue at position 93 among MaSp and minor ampullate spidroin (MiSp) homologs [ 51 , 52 ]. N-terminal domains (NTD) were in some cases also recovered from the RNA-seq analysis ( S2 Fig ).…”

Spider wrapping silk fibre architecture arising from its modular soluble protein precursor

“…To date, only three spidroin repetitive domain structures have been solved 19 20 alongside several non-repetitive N- and C-terminal domain structures 20 21 22 23 24 25 26 . The reported repetitive domain structures are all highly similar seven-helix bundles 19 20 .…”