Liu, P.; Castelino, R. E.; Gierke, T. R.; Wood, A. J.; Lu, Y.; Tang, S.

TMEM95 is a sperm acrosomal membrane protein essential for mammalian fertilization. How TMEM95 facilitates sperm-egg interaction has largely remained unknown. Analogous sperm fertilization proteins function as complexes, leading us to hypothesize that TMEM95 may have a binding partner on sperm. Here, we surveyed interactions between TMEM95 and individual proteins in a curated library of testis-expressed proteins using AlphaFold3. We identify FIMP, a fertilization-essential acrosomal membrane protein, as a high-confidence interaction partner of TMEM95. These two proteins form a high-affinity complex through their ectodomains. Using single-particle cryo-EM, we determine the structure of the human TMEM95-FIMP ectodomain complex at 3.1 angstrom resolution. An aromatic motif of FIMP binds to a conserved surface of TMEM95, and amino acid substitutions within this motif ablate the TMEM95-binding activity of FIMP. We isolate a germline-like anti-TMEM95 antibody, termed 3A02, that binds to human and murine TMEM95 and disrupts the interaction between TMEM95 and FIMP. By determining the cryo-EM structure of human TMEM95 bound to the 3A02 fragment antigen-binding, we find that 3A02 recognizes the FIMP-binding site on TMEM95. An IgM and a single-chain fragment variable of 3A02 inhibit fusion of murine sperm with eggs, independent of antibody size, suggesting that the TMEM95-FIMP interface is critical for sperm-egg interaction. Together, these results establish the human sperm TMEM95-FIMP complex and suggest that a FIMP-mediated interaction of TMEM95 facilitates membrane fusion during mammalian fertilization.