Abstract

Phospholipase C? (PLC?), a sperm-specific enzyme, plays a critical role in mammalian fertilization. Mutations in PLC? have been linked to male infertility, as they impair its ability to trigger calcium (Ca2+) oscillations necessary for egg activation and embryo development. During fertilization, PLC? is introduced into the egg, where it hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate and diacylglycerol, leading to Ca2+ release from the endoplasmic reticulum. Human infertility-associated mutations include H233L, H398P, and R553P, which disrupt PLC? function. To elucidate the molecular consequences of the mutations, we employed full-atom molecular dynamics simulations to analyze structural perturbations and their impact on PIP2 and Ca2+ binding. Our results reveal that H233L and H398P mutations significantly reduce interactions with PIP2, disrupting hydrogen bonding and salt bridge formation, leading to misalignment of the substrate. Additionally, these mutations destabilize Ca2+ binding by altering its positioning within the active site. In contrast, the R553P mutation primarily affects intramolecular stability and enzyme dynamics without impairing substrate or ion binding. Free energy calculations indicate an increased affinity for PIP2 in H233L and H398P mutants, leading to an aberrant substrate positioning and compromised hydrolysis. These structural insights help explain the egg activation failure and infertility of patients carrying these mutations. © 2025 by the authors.