SPACA6 structure reveals a conserved superfamily of gamete fusion-associated proteins

SPACA6 is a sperm-expressed surface protein that is critical for gamete fusion during mammalian sexual reproduction. Despite this fundamental role, little is known about how SPACA6 specifically functions. We elucidated the crystal structure of SPACA6 at 2.2-Å resolution, revealing a two-domain protein containing a four-helix bundle and Ig-like β-sandwich connected via a quasi-flexible linker. Based on the structural analysis, we propose SPACA6 is a founding member of a superfamily of gamete fusion-associated proteins, herein dubbed the IST superfamily. The IST superfamily is defined structurally by its distorted four-helix bundle and a pair of disulfide-bonded CXXC motifs. A structure-based search of the AlphaFold human proteome identified more protein members to this superfamily; remarkably, many of these proteins are linked to gamete fusion. The SPACA6 structure and its connection to other IST-superfamily members provide a missing link in our knowledge of mammalian gamete fusion. Significance Statement SPACA6 is a human sperm protein vital for the fusion of gametes, though its exact function remains a mystery. We present the first solved structure of SPACA6: a two-domain fold comprised of an Ig-like domain and a distorted four-helix bundle. Dali searches of the PDB and AlphaFold reveal a family of structurally related proteins, several of which are also known to play a role in gamete fusion; as such, SPACA6 is a founding member of a conserved protein superfamily, dubbed the IST superfamily. Evolutionary analysis to ascertain functionally relevant structural elements in SPACA6 show a conservation of flexibility between the two domains and several conserved surfaces that could function as protein-protein interfaces.