Mutations that affect ligand binding to the Escherichia coli aspartate receptor - Implications for transmembrane signaling

Three arginine residues of the binding site of the Escherchia coli aspartat e receptor contribute to its high affinity for aspartate (K-d similar to3 m um). Site-directed mutations at residue 64 had the greatest effect on aspar tate binding. No residue could substitute for the native arginine; all chan ges resulted in an apparent K-d of similar to 35 mM. These mutations had li ttle impact on maltose responses. At residue Arg-69, a lysine substitution was least disruptive, conferring an apparent K-d of 0.3 mM for aspartate, R esults obtained for an alanine mutant were similar to those with cysteine a nd histidine mutants (K-d similar to5 mM) indicating that side chain size w as not an important factor here. Proline and aspartate caused more severe d efects, presumably for reasons related to conformation and charge. The impa ct of residue 69 mutations on the maltose response was small. Mutations at Arg-73 had similar effects on aspartate binding (K-d 0.3-7 mm) but more sev ere consequences for maltose responses. Larger side chains resulted in the best aspartate binding, implying steric considerations are important here. Signaling in the mutant proteins was surprisingly robust. Given aspartate b inding, signaling occurred with essentially wild-type efficiency. These res ults were evaluated in the context of available structural data.