Producing positive, negative, and no cooperativity by mutations at a single residue located at the subunit interface in the aspartate receptor of Salmonella typhimurium.
Site-directed mutagenesis of the aspartate receptor of Salmonella typhimurium (Tars) at serine 68, a residue located within the aspartate binding pocket and at the subunit interface, identified this residue as an allosteric switch in this receptor. Substitutions at this position can affect both the type and degree of binding cooperativity observed. Negative cooperativity is observed in the wild-type receptor (nH = 0.7 +/- 0.1) and is maintained by the mutations S68C (nH = 0.8 +/- 0.02), S68V (nH = 0.9 +/- 0.05), and S68D (half-of-the-sites). Binding at only half of the sites was detectable in the S68D mutant, an extreme form of negative cooperativity. No cooperativity (nH = 1.0 +/- 0.03) was observed in the mutant S68A. Positive cooperativity was generated by the substitutions S68T (nH = 1.2 +/- 0.09), S68L (nH = 1.2 +/- 0.1), S68N (nH = 1.3 +/- 0.2), and S68I (nH = 1.4 +/- 0.2). Binding measurements indicated that the substitutions S68Q, S68E, and S68F decrease affinity of the first ligand binding 500-fold, 7000-fold, and 1600-fold, respectively.
Kolodziej
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AF
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Tan
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T
Koshland
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DE
D000494:Allosteric Regulation; D001224:Aspartic Acid / Q000378:metabolism*; D004926:Escherichia coli; D008024:Ligands; D008745:Methylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017459:Receptors, Amino Acid / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction
D008024:Ligands; D017459:Receptors, Amino Acid; D011994:Recombinant Proteins; C031959:aspartic acid receptor; D001224:Aspartic Acid; D012694:Serine