Ultratight Binding of a Capture Spiral Spider Silk Protein through Electrostatic Forces

Web spiders assemble soluble silk proteins, so-called spidroins, into fibers with extraordinary toughness. The spidroin N-terminal domains (NTDs) are highly conserved self-assembly modules that dimerize induced by chemical stimuli within the spinning duct. Spidroins from the flagelliform spinning gland form tough and elastic capture spiral silk. Their NTDs exhibit an unusual content of charged amino acid side chains with an unknown function. Here, we engineered a molecular exciton probe into a flagelliform spidroin NTD to detect the mechanism and strength of binding through kinetic fluorescence in combination with chemical denaturation experiments. Site-directed mutagenesis experiments revealed that salt bridges formed by few additional side chain charges tighten the dimer dramatically, reducing the equilibrium dissociation constant by five to six orders of magnitude. The central helix of the dimerization interface resembles a dipolar electrostatic stalk that, through cooperative effects within an antiparallel dimer, generates an equilibrium dissociation constant in the femtomolar range. The additional electrostatic forces acting between flagelliform spidroin NTDs are likely to contribute to the remarkable mechanical properties of capture spiral silk.