Structural features, intrinsic disorder, and modularity of a pyriform spidroin 1 core repetitive domain

Abstract

Orb-weaving spiders produce up to seven silk types, each with distinct biological roles, protein compositions and mechanics. Pyriform (or piriform) silk is composed of pyriform spidroin 1 (PySp1) and is the fibrillar component of attachment discs that attach webs to substrates and to each other. Here, we characterize the 234-residue repeat unit (the “Py unit”) from the core repetitive domain of <i>Argiope argentata</i> PySp1. Solution-state nuclear magnetic resonance (NMR) spectroscopy-based backbone chemical shift and dynamics analysis demonstrates a structured core flanked by disordered tails, structuring that is maintained in a tandem protein of two connected Py units, indicative of structural modularity of the Py unit in the context of the repetitive domain. Notably, AlphaFold2 predicts the Py unit structure with low confidence, echoing low confidence and poor agreement to the NMR-derived structure for the <i>Argiope trifasciata</i> aciniform spidroin (AcSp1) repeat unit. Rational truncation, validated through NMR spectroscopy, provided a 144-residue construct retaining the Py unit core fold, enabling near-complete backbone and side-chain <sup>1</sup>H, <sup>13</sup>C and <sup>15</sup>N resonance assignment. A six α-helix globular core is inferred, flanked by regions of intrinsic disorder that would link helical bundles in tandem repeat proteins in a beads-on-a-string architecture.

Physical Properties of the Second Type of Aciniform Spidroin (AcSp2) from Neoscona theisi Reveal a pH-Dependent Self-Assembly Repetitive Domain

“…Like AcSp repeat domains, the TuSp1 and PySp1 repeat domains have seven-helix and six αhelix globular cores, respectively, which form a beads-on-astring architecture. 45,46 Despite the lack of information for tertiary structures, the secondary structures of all three N. theisi AcSp repetitive domains in neutral solution also exhibit mainly an α-helical structure, suggesting that the structures of AcSp repeat domains within and among spider species are similar. Based on these findings and our results, therefore, it is possible that the repetitive domains of AcSp, TuSp, and PySp in neutral solution have evolved similar structures despite different amino acid sequences.…”

“…47 In general, pH-dependent self-assembly domains are found in terminal regions that are responsible for silk formation, while repeat domains determine the mechanical properties of silk fibers. 10,46 Given the low sequence similarity and different self-assembly capacities between AcSp1 and AcSp2, it is probable that AcSp2 has distinct mechanical properties and functions from AcSp1 proteins. The mechanical properties of spider silks are largely dependent on the spidroin repetitive domain.…”

“…In the previous studies, the soluble form of Argiope trifasciata AcSp1 repetitive domain is proved to contain 5 helical segments and is connected to each other by disordered regions, and the solution structure of Nephila antipodiana AcSp1 repeat unit has a seven-helix bundle. , Additionally, these helix-rich structures are not only found in AcSp repeat units but also exist in other spidroin types. Like AcSp repeat domains, the TuSp1 and PySp1 repeat domains have seven-helix and six α-helix globular cores, respectively, which form a beads-on-a-string architecture. , Despite the lack of information for tertiary structures, the secondary structures of all three N. theisi AcSp repetitive domains in neutral solution also exhibit mainly an α-helical structure, suggesting that the structures of AcSp repeat domains within and among spider species are similar.…”

“…Recently, the MiSp repetitive domain has also been found to have pH-sensitive aggregation property, which is proved to be caused by charge-dipole interactions . In general, pH-dependent self-assembly domains are found in terminal regions that are responsible for silk formation, while repeat domains determine the mechanical properties of silk fibers. , Given the low sequence similarity and different self-assembly capacities between AcSp1 and AcSp2, it is probable that AcSp2 has distinct mechanical properties and functions from AcSp1 proteins.…”

A recombinant chimeric spider pyriform-aciniform silk with highly tunable mechanical performance

“…Mechanics of Py 2 -based fibers were observed to be more readily tunable than W-based fibers through relatively modest changes in post-spin stretching conditions, but fibers wet-spun from this protein construct are not amenable to post-spin stretching in water [ 3 ]. Both the W unit and Py unit exhibit mixed α-helical and disordered secondary structure in the solution-state [ 27 , 30 ], with partial loss of α-helical content and appearance of β-sheet structuring in the fiber states of both recombinant spidroins [ 3 , 27 ]. These structural transformations, including a retention of α-helical character in the fibrous states, are akin to those observed in natural aciniform and pyriform silks [ 31 ].…”