Abstract
Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct.
Highlights
Manner within the duct[25,26,27]
Throughout 34 so far analysed sequences of amino-terminal domains from different types of spidroins or spider species, amino acid residues D39, E76, E81 and E114 are the most conserved ones[24]. These residues were replaced in the amino-terminal domain (NRN1L.h.) of MaSp1 of the black widow spider (Latrodectus hesperus) by the non-titratable analogues asparagine and glutamine (Fig. 1a) to investigate their contribution to dimerization and conformational conversion
The pH-dependent conformational state of wtNRN1L.h. was determined by far- and near-UV circular dichroism (CD) spectroscopy revealing a significant pH-dependence of tertiary (Fig. 1b–e) but not of secondary structure (Supplementary Fig. S3)
Summary
Manner within the duct[25,26,27]. The previously determined structure of Euprosthenops australis (NRN1E.a.) as well as of Latrodectus hesperus (NRN1L.h.) MaSp1 revealed a dipolar charge distribution, with charged amino acids being grouped into a basic NH2- and an acidic COOH-terminal pole[26,28,29]. The conformational change was monitored by a bathochromic shift of the fluorescence emission of a single, naturally occurring Trp residue (W9) indicating its relocation into a more hydrophilic environment on the surface[12,28]. Underlining their likely importance for this conformational change, one aspartic acid (D39) and three glutamic acid residues (E76, E81, E114) are the most conserved residues within NRN among all spider species so far investigated[20,21,22,23,24]. Dimerization and structural rearrangement occur independently and are controlled by separated regions of the domain
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