Spidroin N-terminal Domain Promotes a pH-dependent Association of Silk Proteins during Self-assembly

Abstract

Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD.

Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation

“…The spectra recorded at pH 8.0 show a typical behaviour for wt NT 13,25 , a mainly a-helical secondary structure at 25°C and random-coil structure at 95°C, which is able to refold upon cooling (Fig. 4a).…”

“…The results are expressed as the ratio between the fluorescence at 339 and 351 nm, which reflects the ratio between monomeric and dimeric NT 16 . Since the presence of NaCl stabilizes the monomer conformation 13,14 , measurements were made without salt as well as at a physiological concentration (154 mM). Similar shifts in Trp fluorescence ratios were observed when NaCl was substituted with Na 2 SO 4 or NaNO 3 ( Supplementary Fig.…”

“…NT is mainly monomeric at pH 7 and dimeric at pH 6 (refs [13][14][15] but the associated molecular mechanisms are still largely unknown. The high-resolution X-ray structure of MaSp1 NT from Euprosthenops australis demonstrates a homodimer, where each subunit adopts a fivehelix bundle fold and helix 2, 3 and 5 form the dimer interface.…”

“…The MaSp1 NT domain is highly sensitive to changes in pH and is proposed to function as a pH-regulated relay with a dual function: conferring solubility at high pH and facilitating fibre formation at low pH [12][13][14] . NT is mainly monomeric at pH 7 and dimeric at pH 6 (refs [13][14][15] but the associated molecular mechanisms are still largely unknown.…”

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

“…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. 28,36 The presence of the NRN domain in engineered, recombinant spider silk proteins decelerates the rate of aggregation by more than one order of magnitude, indicating a stabilizing role during storage. 26 Acidification to a pH below 6.4, which naturally occurs during the spinning process, induces the formation of a tight antiparallel NRN dimer, probably acting as an intermolecular crosslinker.…”