Interaction of α-Agglutinin and a -Agglutinin, Saccharomyces cerevisiae Sexual Cell Adhesion Molecules

Abstract

␣-Agglutinin and a-agglutinin are complementary cell adhesion glycoproteins active during mating in the yeast Saccharomyces cerevisiae. They bind with high affinity and high specificity: cells of opposite mating types are irreversibly bound by a few pairs of agglutinins. Equilibrium and surface plasmon resonance kinetic analyses showed that the purified binding region of ␣-agglutinin interacted similarly with purified a-agglutinin and with a-agglutinin expressed on cell surfaces. At 20°C, the K D for the interaction was 2 ؋ 10 ؊9 to 5 ؋ 10 ؊9M. This high affinity was a result of a very low dissociation rate (Ϸ 2.6 ؋ 10 ؊4 s ؊1 ) coupled with a low association rate ‫؍(‬ 5 ؋ 10 4 M ؊1 s ؊1 ). Circular-dichroism spectroscopy showed that binding of the proteins was accompanied by measurable changes in secondary structure. Furthermore, when binding was assessed at 10°C, the association kinetics were sigmoidal, with a very low initial rate. An induced-fit model of binding with substantial apposition of hydrophobic surfaces on the two ligands can explain the observed affinity, kinetics, and specificity and the conformational effects of the binding reaction.

A Biochemical Guide to Yeast Adhesins: Glycoproteins for Social and Antisocial Occasions

“…Because both ␣-agglutinin and its ligand a-agglutinin have been extensively characterized, we can describe a model of the binding. Binding of ␣-agglutinin to high-affinity peptide epitopes on the a-agglutinin binding subunit Aga2p is slow and tight (K D of ϳ10 Ϫ9 M) (10,106,141). The agglutinins are displayed on the outer surface of the cell wall (10, 133) at 10 4 to 5 ϫ 10 4 copies per cell, corresponding to concentrations of 10 to 100 M in the 100-nm space between the cells (Fig.…”

“…In addition, conformational shifts in the agglutinins are essential for effective binding, even in this well-characterized, apparently simple reaction (141). Binding of the S. cerevisiae agglutinins is kinetically irreversible, due to very low dissociation rates (hours to days) (80,141).…”

“…In contrast, in vitro binding assays such as surface plasmon resonance or 125 I-protein binding assays can document only much tighter interactions, with affinities in the nanomolar to micromolar range. Indeed, the agglutinins bind with both nanomolar and micromolar affinities (106,141). Because the C-terminal residues of Aga2p are the major binding determinant, a surface display model that fuses peptides to be screened to the C terminus of Aga2p inactivates the high-affinity binding (106,129).…”

Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

“…The polysaccharides that provide the mechanical strength of the cell wall also serve as the attachment matrix for a wide variety of glycoproteins (Zlotnick et al 1984;Klis et al 2006). These glycoproteins include sexual agglutination factors important for mating (Cappellaro et al 1994;Zhao et al 2001) and adhesins critical to cell-cell contact during filamentation, invasive growth, and biofilm formation (Reynolds and Fink 2001;Douglas et al 2007). Cellsurface glycoproteins also limit the permeability of the cell wall to macromolecules, thereby protecting the glucan layer from wall-degrading enzymes (Zlotnik et al 1984;De Nobel et al 1990;De Nobel and Barnett 1991;Klis et al 2006).…”

Delineation of Functional Regions within the Subunits of theSaccharomyces cerevisiae Cell Adhesion Molecule a-Agglutinin

“…Cys residues at positions 7 and 50 of mature Aga2p (corresponding to positions 25 and 68 of the open reading frame) anchor it to the cell surface through Aga1p; both cysteine residues must be mutated to lose cellular agglutinability (20). Aga2p is necessary and sufficient for binding to ␣-agglutinin, and binding is tight and practically irreversible over the time course of a mating reaction (21,44). Synthetic or proteolytic peptides containing the ten C-terminal residues of Aga2p are sufficient to bind ␣-agglutinin, but at concentrations 4-to 5-fold higher than the full-length peptide (20), indicating that this region has a major binding determinant and that carbohydrate or * Supported by the Grants 1R01-GM47176 and 2SO6-GM60654 from NIGMS, National Institute of Health (NIH) and by Grant RR-03037 from the Research Centers in Minority Institutions Program of NIH.…”