title: Two-state allosteric modeling suggests protein equilibrium as an integral component for cyclic AMP (cAMP) specificity in the cAMP receptor protein of Escherichia coli.
abstract: Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 A) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that wild-type CRP should be activated even by the lower-affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues.
authors.affiliation: Department of Bacteriology, 1550 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Hwan
authors.initials: H
authors.lastname: Youn
authors.forename: Junseock
authors.initials: J
authors.lastname: Koh
authors.forename: Gary P
authors.initials: GP
authors.lastname: Roberts
mesh_terms: D000241:Adenosine / Q000378:metabolism; D000494:Allosteric Regulation; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D008962:Models, Theoretical; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D002373:Cyclic AMP Receptor Protein; D000242:Cyclic AMP; D006152:Cyclic GMP; D000241:Adenosine