Structure-guided analysis of divergent homologs unveils deep ancestry and arthropod specialization of the pacifastin family

Abstract The Pacifastin family has been widely regarded as an arthropod-specific innovation, functionally restricted to the regulation of innate immunity. Here, we challenge this paradigm using a structural phylogenomics approach that overcomes the sequence erosion characteristic of small disulfide-rich proteins. We identified a population of “cryptic” homologues comprising ~ 30% of the dataset—undetectable by sequence alone and demonstrated that the characteristic Pacifastin’s six-cysteine scaffold is not an arthropod invention but an ancient lineage whose evolutionary roots extend across major metazoan phyla, Fungi, and Bacteria. Structural and architectural profiling revealed two recurrent organizational modes: an ancestral class characterized by an N-terminal β-hairpin extension, glycine-rich and frequently embedded in multidomain, extracellular matrix–associated proteins, and a conventional, derived arthropod-restricted class characterized by modular simplification and predicted structural rigidity consistent with a soluble-effector function. We propose a "Liberation and Structural Convergence" evolutionary model to explain this transition: the acquisition of proteolytic processing sites in inter-domain linkers is consistent with a mechanism by which arthropods may have released these domains from the matrix, while the concurrent structural convergence is consistent with adaptation to a soluble, circulating hemolymph environment. Further phylogenomic profiling of the reactive site revealed a functional shift from an ancestral inhibitory signature enriched in basic and charged residues to a derived arthropod-specific radiation toward hydrophobic residues. This transition suggests a broadening of target specificity toward chymotrypsin- and elastase-like proteases, consistent with the recruitment of these inhibitors into arthropod immune roles. Together, these results reposition pacifastins as an ancient protein lineage and illustrate how modularity, proteolytic processing, and biophysical constraints may have driven the transition from matrix-embedded regulatory scaffolds to systemic soluble effectors.