Background: The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 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. Results: We made an in silico 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 spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 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 C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants. Conclusions: The results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans 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 C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain.
Bibliographical noteFunding Information:
We thank Matt Andrews and Shannon Stevenson for critical comments on the manuscript. We also thank Julia Curry and Kayla York for performing preliminary analysis of some mutant transgenes. Some worm strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. The spe-42(tm2421) allele was provided by S. Mitani at the Japanese National BioResource Project http:// www.shigen.nig.ac.jp/c.elegans/index.jsp. Funding to TLK was from the National Science Foundation (IOS-0918464) under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5), University of Minnesota Startup Funds, and a University of Minnesota Faculty Grant-in-Aid.