Self-assembly of spider silk proteins is controlled by a pH-sensitive relay

Abstract

Nature's high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider's spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 A resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider's silk extrusion duct.

The role of terminal domains during storage and assembly of spider silk proteins

“…25 However, several results based on various sequence comparisons together with structural data of different silk types strongly indicate that the non-repetitive amino-terminal domain is also present in the mature fiber. 20,22,26 A significant step toward understanding the role of the terminal domains in storage and assembly was the determination of atomistic structures of the non-repetitive terminal regions of MaSp proteins. Interestingly, both terminal domains are mainly composed of unique a-helix-bundles but with different folds, neither of which have structural homologues so far.…”

“…Interestingly, both terminal domains are mainly composed of unique a-helix-bundles but with different folds, neither of which have structural homologues so far. [26][27][28] The MaSp NRN domains from Euprosthenops australis and Latrodectus hesperus (black widow) consist of five a-heli- ces and are monomeric at pH 6.8 and above. On acidification they form tight, antiparallel dimers ( Figures 3A and 3B).…”

“…The dimer interface is mainly hydrophobic and conserved throughout different species and silk types. 26 The dimer reveals an interesting charge distribution with positive and negative charges clustering in opposite directions, forming a macromolecular dipole. Modeling experiments showed that this feature seems to be conserved in different NRN domains from different species and silk types.…”

“…The crystal structure of NRN of E. australis revealed that the linker region of one subunit connecting NRN with the repetitive core domain fits into this crevice, probably acting as a structural lock. 26 The NRC domain of the MaSp2 analog ADF3 (Araneus diadematus fibroin 3) from A. diadematus (European garden spider) also consists of five a-helices ( Figure 3C). In contrast to the amino-terminal domain, the NRC region is a permanent, parallel dimer, covalently linked by a disulfide bond.…”

“…Recent results, supported by the atomic resolution structures of MaSp-termini (see above), indicate that both terminal domains act as environmentally triggered structural switches significantly influencing the solubility as well as the initiation of assembly. [26][27][28]35 Both MaSp1 amino-terminal regions, one from Euprosthenops australis and one from L. hesperus (black widow), are monomeric at storage conditions (pH 7 in presence of sodium chloride). In the complete absence of salt, the NRN domain has a slight tendency to dimerize, which is fully suppressed in the presence of 300 mM sodium chloride.…”