Evidence from Flagelliform Silk cDNA for the Structural Basis of Elasticity and Modular Nature of Spider Silks Cheryl Y. Hayashi* and Randolph V. Lewis Department of Molecular Biology, University of Wyoming, Laramie WY 82071-3944, USA Orb-web weaving spiders rely on their aerial nets to entrap ¯ying prey. A key mechanical feature of orb-web design is the high elasticity of the capture spiral. We report the cloning of substantial cDNA for ¯agelliform gland silk protein, which forms the core ®ber of the catching spiral. Like all silks, the ¯agelliform protein is composed largely of iterated sequences. The dominant repeat of this protein is Gly-Pro-Gly-Gly-X, which can appear up to 63 times in tandem arrays. This motif likely forms Pro 2 -Gly 3 type II b-turns and the resulting series of concatenated b-turns are thought to form a b-spiral. We propose that this spring-like helix is the basis for the elasticity of silk. The variable ®fth position of the motif (X) is occupied by a small subset of residues (Ala, Ser, Tyr, Val). Moreover, these X amino acids occur in speci®c patterns throughout the repeats. This ordered variation strongly suggests that with hydration, the b-spirals form hydrogen-bonded networks that increase the elasticity of ¯agelliform silk. The self-assembly of ¯agelliform protein monomers into silk ®bers may be promoted by b-spiral/b-spiral interactions. Addition- ally, the other two motifs in the ¯agelliform protein, Gly-Gly-X and a spacer that disrupts the glycine-rich regions, may contribute to the align- ment of monomers into ®bers. The ¯agelliform protein cDNA was compared to the other members of the spider silk gene family. We show that all spider silk proteins can be characterized as sets of shared structural modules. The occurrence of these modules among the proteins is inconsistent with the phylogenetic relationships inferred from the C-terminal regions. This observation, along with the high level of variation among individual ¯agelliform pro- tein repeats, but striking lack of such variation in the other silk proteins, suggests that unusual homogenization processes are involved in silk pro- tein evolution. # 1998 Academic Press Limited Keywords: elasticity; ¯agelliform; protein; silk; spider *Corresponding author Introduction Spider silks are an ideal system for studying the relationship between protein design and function. While silk production has evolved multiple times within arthropods, silk use is most highly devel- oped in spiders. Spiders are unique in their ability to spin an array of silk proteins throughout their lifetimes. Each type of silk is secreted and stored by a different type of abdominal gland until extruded by tiny spigots on the spinnerets. These Abbreviations used: silk genes from Nephila clavipes: Flag, ¯agelliform ®broin; MaSp1, major ampullate spidroin 1 (Xu & Lewis, 1990); MaSp2, major ampullate spidroin 2 (Hinman & Lewis, 1992). Silk genes from Araneus diadematus: ADF-1, ®broin 1; ADF-2, ®broin 2; ADF-3, ®broin 3; ADF-4, ®broin 4 (all Guerette et al., 1996); ABF-1, C-terminal sequence from Araneus bicentenarius (Beckwitt & Arcidiacono, 1994). Phylogenetic tree descriptors: CI, consistency index excluding uninformative characters (Kluge & Farris, 1969); RI, retention index (Farris, 1989). J. Mol. Biol. (1998) 275, 773±784 0022±2836/98/050773±12 $25.00/0/mb971478 # 1998 Academic Press Limited