Molecular evolutionary analysis of nematode Zona Pellucida (ZP) modules reveals disulfide-bond reshuffling and standalone ZP-C domains

Zona pellucida (ZP) modules mediate extracellular protein-protein interactions and contribute to important biological processes including syngamy and cellular morphogenesis. While some biomedically-relevant ZP modules are well-studied, little is known about the protein family’s broad-scale diversity and evolution. The increasing availability of sequenced genomes from “non-model” systems provides a valuable opportunity to address this issue, and to use comparative approaches to gain new insights into ZP module biology. Here, through phylogenetic and structural exploration of ZP module diversity across the nematode phylum, I report evidence that speaks to two important aspects of ZP module biology. First, I show that ZP-C domains—which in some modules act as regulators of ZP-N domain-mediated polymerization activity, and which have never before been found in isolation—can indeed be found as standalone domains. These standalone ZP-C domain proteins originated in independent (paralogous) lineages prior to the diversification of extant nematodes, after which they evolved under strong stabilizing selection, suggesting the presence of ZP-N domain-independent functionality. Second, I provide a much-needed phylogenetic perspective on disulfide bond variability, uncovering evidence for both convergent evolution and disulfide-bond reshuffling. This result has implications for our evolutionary understanding and classification of ZP module structural diversity and highlights the usefulness of phylogenetics and diverse sampling for protein structural biology. All told, these findings set the stage for broad-scale (cross-phyla) evolutionary analysis of ZP modules and position Caenorhabditis elegans and other nematodes as important experimental systems for exploring the evolution of ZP modules and their constituent domains.

透明带（zona pellucida, ZP）结构域介导细胞外蛋白质-蛋白质相互作用，并参与包括配子配合与细胞形态发生在内的重要生物学过程。尽管部分与生物医学相关的ZP结构域已得到充分研究，但学界对该蛋白质家族的整体多样性与演化机制仍知之甚少。近年来，“非模式生物”测序基因组的日益丰富为解决这一问题提供了宝贵契机，也使得我们能够通过比较研究方法，为ZP结构域生物学研究带来新的见解。本文通过对线虫门类群的ZP结构域多样性开展系统发育与结构层面的探究，揭示了与ZP结构域生物学相关的两项重要发现。其一，研究证实，ZP-C结构域（ZP-C domain）——在部分ZP结构域中可调控ZP-N结构域（ZP-N domain）介导的聚合活性，且此前从未被发现以独立形式存在——确实可以作为独立结构域单独存在。这类独立ZP-C结构域蛋白起源于现存线虫类群分化前的独立旁系同源谱系，后续经历了强烈的稳定选择演化，提示其存在不依赖ZP-N结构域的独特生物学功能。其二，本研究为ZP结构域的二硫键变异提供了亟需的系统发育视角，同时发现了趋同演化与二硫键重排的相关证据。这一结果有助于深化我们对ZP结构域结构多样性的演化理解与分类体系完善，同时凸显了系统发育分析与多样化采样在蛋白质结构生物学研究中的重要价值。综上，本研究为ZP结构域的跨门类大规模演化分析奠定了基础，并确立了秀丽隐杆线虫（Caenorhabditis elegans）与其他线虫作为重要实验系统的地位，用于探索ZP结构域及其组成结构域的演化历程。