Simmons Jeffrey R, Gasmi-Seabrook Geneviève, Rainey Jan K
Department of Biochemistry& Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada.
Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2M9, Canada.
Biochem Cell Biol. 2023 Aug 1;101(4):271-283. doi: 10.1139/bcb-2022-0338. Epub 2023 Feb 21.
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 PySp1. Solution-state nuclear magnetic resonance (NMR) spectroscopy-based backbone chemical shift and dynamics analysis demonstrate 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 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 H, C, and 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.
圆网蛛能产生多达七种丝，每种丝都有独特的生物学作用、蛋白质组成和力学性能。梨形丝由梨形蛛丝蛋白1（PySp1）组成，是将蛛网固定在基质上以及蛛网各部分之间相连的附着盘的纤维成分。在此，我们对PySp1核心重复结构域中的234个残基重复单元（“Py单元”）进行了表征。基于溶液态核磁共振（NMR）光谱的主链化学位移和动力学分析表明，其核心结构有序，两侧为无序的尾部，这种结构在由两个相连的Py单元组成的串联蛋白中得以保持，这表明在重复结构域的背景下，Py单元具有结构模块性。值得注意的是，AlphaFold2对Py单元结构的预测可信度较低，这与对aciniform蛛丝蛋白（AcSp1）重复单元的NMR衍生结构的低可信度和较差的一致性相呼应。通过NMR光谱验证的合理截短，得到了一个保留Py单元核心折叠的144个残基的构建体，实现了近乎完整的主链和侧链H、C和N共振归属。由此推断出一个由六个α螺旋组成的球状核心，两侧是内在无序区域，这些区域将以串珠状结构连接串联重复蛋白中的螺旋束。
