Abstract
Spider silk is self-assembled from water-soluble silk proteins through changes in the environment, including pH, salt concentrations, and shear force. The N-terminal domains of major and minor ampullate silk proteins have been found to play an important role in the assembly process through salt- and pH-dependent dimerization. Here, we identified the sequences of the N-terminal domains of aciniform silk protein (AcSpN) and major ampullate silk protein (MaSpN) from Nephila antipodiana (NA). Different from MaSpN, our biophysical characterization indicated that AcSpN assembles to form large oligomers, instead of a dimer, upon condition changes from neutral to acidic pH and/or from a high to low salt concentration. Our structural studies, by nuclear magnetic resonance spectroscopy and homology modelling, revealed that AcSpN and MaSpN monomers adopt similar overall structures, but have very different charge distributions contributing to the differential self-association features. The intermolecular interaction interfaces for AcSp oligomers were identified using hydrogen–deuterium exchange mass spectrometry and mutagenesis. On the basis of the monomeric structure and identified interfaces, the oligomeric structures of AcSpN were modelled. The structural information obtained will facilitate an understanding of silk fiber formation mechanisms for aciniform silk protein.
Highlights
Silk production from the opisthosoma glands is the most diagnostic apomorphy of the spider
According to pairwise sequence alignment, aciniform silk protein (AcSpN) from Nephila antipodiana (NA) shares 95% and 58% amino acid sequence identities with AcSpNs from Trichonephila clavipes (TC) and Argiope trifasciata (AT), respectively, but it shares only 34–37% sequence identities with the major ampullate silk protein (MaSpN) from NA, E. australis (EA), and N. clavipes (NC)
The results show that N-terminal domain (NTD) share high sequence identities within the same type of silk proteins from different spider species, Int
Summary
Silk production from the opisthosoma glands is the most diagnostic apomorphy of the spider. To make spider silk available on a large scale, various recombinant methods have been attempted, but with limited success, due to a poor understanding of the molecular mechanisms of silk fiber formation and silk structure–property relationships [6,7,8,9,10]. Spider silk is an orderly assembly of one or more types of spider silk proteins or spidroins. The C-terminal domain (CTD) is relatively conserved among different types of silk proteins [17] and plays an important role in maintaining a spidroin in a solution-competent form by dimerization [18,19,20]. The CTD reduces unspecific aggregation of a spidroin during its storage and ensures the correct alignment of the RP domains to form a well-defined fiber assembly during fiber formation [18,20]
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