The molecular nature of the F-actin binding activity of aldolase revealed with site-directed mutants.

We used site-directed mutagenesis of rabbit muscle aldolase, falling ball viscometry, co-sedimentation binding assays, and negative stain electron microscopy, to identify specific residues involved in the aldolase-actin interaction. Three mutants, R42A (Arg --> Ala), K107A (Lys --> Ala), and R148A (Arg --> Ala), had minimal actin binding activity relative to wild type (wt) aldolase, and one mutant, K229A (Lys --> Ala), had intermediate actin binding activity. A mutant with approximately 4,000-fold reduced catalytic activity, D33S (Asp --> Ser), had normal actin binding activity. The aldolase substrates and product, fructose 1,6-bisphosphate, fructose 1-phosphate, and dihydroxyacetone phosphate, reversed the gelling of wt aldolase and F-actin, consistent with at least partial overlap of catalytic and actin-binding sites on aldolase. Molecular modeling reveals that the actin-binding residues we have identified are clustered in or around the catalytic pocket of the molecule. These data confirm that the aldolase-actin interaction is due to specific binding, and they suggest that electrostatic interactions between specific residues, rather than net charge, mediate this interaction. Low concentration of wt and D33S aldolase caused formation of high viscosity actin gel networks, while high concentrations of wt and D33S aldolase resulted in solation of the gel by bundling actin filaments, consistent with a potential role for this enzyme in the regulation of cytoplasmic structure.