Moonlighting Proteins: Some Hypotheses on the Structural Origin of Their Multifunctionality

Moonlighting proteins—single polypeptides performing multiple, often unrelated functions—are increasingly recognized as key players in human disease and microbial pathogenesis, making their identification crucial for understanding disease mechanisms and developing targeted therapies. This study addresses the unresolved question of how such multifunctionality evolves, focusing on two potential structural mechanisms: Non-Orthologous Gene Displacement/Non-Homologous Isofunctional Enzymes (NOGD/NHIE), where evolutionarily unrelated proteins perform the same function, and Fold-Switching Proteins (FSP), which adopt alternative secondary structures to switch functions without sequence changes. We analyzed the overlap between known human moonlighting proteins (from MultitaskProtDB-II) and curated datasets of NOGD/NHIE (Non-Orthologous Gene Displacement/Non-Homologous Isofunctional Enzymes) and fold-switching proteins (FSPs), using Fisher’s exact test for statistical validation. Moonlighting proteins showed extraordinary enrichment for NOGD/NHIE (19.89% vs. 0.39% in non-moonlighting proteins; odds ratio = 63.1, p < 2.2 × 10−16) and strong enrichment for FSPs (6.99% vs. 0.26%; odds ratio = 28.7, p = 1.13 × 10−14), corresponding to ~51-fold and ~27-fold higher risks, respectively. These findings establish intrinsic structural plasticity—whether through evolutionary replacement (NOGD/NHIE) or conformational switching (FSP)—as a central mechanism enabling functional moonlighting in the human proteome. The results suggest that such plasticity facilitates functional innovation while preserving sequence integrity, and that both NOGD/NHIE and FSP features may serve as predictive signatures for identifying novel moonlighting proteins, particularly those with implications for disease mechanisms and therapeutic targeting.