Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>The<jats:italic>C. elegans</jats:italic>sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with<jats:italic>spe-42</jats:italic>mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>We made an<jats:italic>in silico</jats:italic>structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created<jats:italic>spe-42</jats:italic>transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn<jats:sup>++</jats:sup>ions in the RING finger motif. Transgenes were crossed into a<jats:italic>spe-42</jats:italic>null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in<jats:italic>C. elegans</jats:italic>and<jats:italic>C. briggsae</jats:italic>SPE-42 following the RING finger domain are not responsible for the failure of the<jats:italic>C. briggsae</jats:italic>SPE-42 homolog to rescue<jats:italic>C. elegans spe-42</jats:italic>mutants.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>The results suggest that a<jats:italic>bona fide</jats:italic>RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during<jats:italic>C. elegans</jats:italic>fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the<jats:italic>C. elegans</jats:italic>and<jats:italic>C. briggsae</jats:italic>SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain.</jats:p></jats:sec>