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

Abstract

The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface

“…This provides a mechanism for plasticity in the intermolecular contacts at the dimer interface that could be relevant during the transition from loosely to stably associated dimer. The fact that mutation of Asp 39 abolishes NTD dimerization is consistent with the idea that structural plasticity plays a role in dimerization, as Asp 39 engages in a key short-range salt bridge in our Nc NTD structure but not in the more symmetric Ea NTD structure (13,16 The electrostatic potentials were calculated using the Poisson-Boltzmann method as implemented in CCP4mg with red and blue representing negative and positive electrostatic potential, respectively, on a scale ranging from ϩ0.5 V to Ϫ0.5 V. C, chain Z (green) was built as a Ser-Tyr-Gly tripeptide into an area of electron density between subunit B of the Nc NTD (orange) in the asymmetric unit and subunit B.x of a symmetry-related NTD molecule (blue). This tripeptide corresponds to C-terminal residues 136 -138 of the Nc NTD.…”

“…2C). Mutation of each of these residues leads to a decrease in dimer formation, confirming their involvement in dimerization (12,13,16 (Fig. 2A, top row, right).…”

“…Among these asymmetric interactions are novel intermolecular salt bridges at opposite ends of the dimer (Asp 39 -Lys 65 and Asp 40 -Lys 65 ) that provide new insights into the crucial role these residues play in NTD dimer formation. Our structure also reveals a unique intramolecular "handshake" interaction between Asp 17 and Asp 53 , which contributes to the asymmetry seen in our structure and potentially adds an additional layer of complexity to the pH-sensitive relay mechanism for NTD dimerization proposed by Knight and colleagues (11,13). We investigated the role these key salt bridge and handshake interactions play in dimer formation using a tryptophan fluorescence assay, and the results support our structural observations.…”

“…Glutamate 84, which engages in the Asp 40 -Glu 84 handshake interaction, is known to be a key player in the pH-dependent relay mechanism that controls NTD dimerization (13). The proximity of negatively charged carboxylates can lead to a substantial elevation of side chain pK a to a physiological value.…”

“…A concomitant decrease in pH to ϳ6.5 results in protonation of two acidic residues (Glu 79 and Glu 119 ) (12), which promotes structural conversions within the NTD subunits that are also required to form this weakly associated dimer. As the pH decreases to ϳ5.7 near the end of the spinning duct, protonation of a third acidic residue (Glu 84 ) facilitates formation of the fully stable dimer (13). Notably, all of the key interactions at the dimer interface exhibit a high degree of symmetry, meaning that equivalent residues of each subunit engage in the same sets of contacts (11).…”