Understanding mammalian fertilization at the molecular level: Investigations of the fertilin beta-alpha(6)beta(1) integrin interaction

Binding of a sperm to the egg plasma membrane mediated through protein-protein interactions between both cell surfaces is an essential step leading to mammalian fertilization. Fertilinβ is present on the equatorial membrane of fertilization competent sperm in all mammalian species. It is clear from a combination of photoaffinity labeling experiments and cell-binding assays with recombinant wild-type and mutant proteins that fertilinβ binds directly to α ₆β₁ integrin on egg surface. More specifically, this interaction is mediated through the disintegrin domain of fertilinβ. A conserved binding motif, ECD, of the fertilinβ disintegrin domain is the sequence recognized by α₆β₁ integrin. However, nothing was known about the structure of the fertilinβ binding pocket in α₆β₁, because no structural information was available for either fertilinβ or integrin α₆β ₁. In this work, the structural requirements for this protein-protein interaction were explored from two perspectives: (a) The required conformation of the sperm surface protein fertilinβ required for receptor binding, was determined using conformational analysis of cyclic peptide analogs. (b) The identity of the ligand binding site for fertilinβ in the egg membrane receptor α₆β₁ integrin was probed by proteolytic mapping of the receptor tagged with a photoaffinity ligand.; A series of cyclic and linear peptides containing the conserved (D)ECD sequence from the disintegrin domain fertilinβ were synthesized and tested as inhibitors of sperm-egg fusion in in vitro mouse fertilization assays. NMR data were combined with computer aided conformational searching to determine which low energy conformer(s) predominate in solution. We found one possibility for the high IC₅₀'s of these (D)ECD cyclic peptides is that the acidic residues are not constrained to a sufficiently tight turn. To determine the binding site on the egg receptor α₆β ₁ integrin for the inhibitory DECD peptides, and thus fertilinβ, a photoaffinity labeled peptide was synthesized and utilized in receptor crosslinking experiments. After exploring various combinations of purification, isolation, and identification, we developed a protocol using a combination of conjugate proteolysis, followed by immobilized avidin purification, and MALDI-TOF spectrometric identification and sequencing to map the ligand binding site.