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.

FIG. 1.

Shared activation properties of the CRP* variants. (A) The in vitro DNA affinity of the CRP proteins in the absence of ligand was measured by fluorescence anisotropy at protein levels of 100 nM. The anisotropy values for “no DNA binding” and for “saturated DNA binding” were 0.138 and 0.181 (WT CRP + 100 μM cAMP), respectively. Each CRP* substitution was also made in T127L or S128I CRP backgrounds, and the DNA affinity was also measured as follows: left panel, WT and CRP* variants; center panel, WT and CRP* variants with T127L; right panel, WT and CRP* variants with S128I. (B) The CRP* variants were activated by various ligands, while WT CRP is activated only by cAMP. The DNA affinity of each protein (at 100 nM) was measured by a fluorescence anisotropy method, but with various ligands: 0.1 mM cAMP, 0.1 mM cGMP, 0.4 mM adenosine, and 4 mM AMP.

FIG. 2.

Models of the equilibrium of CRP with or without ligands. (A) Simple equilibrium-shift model. CRP exists in an equilibrium between active (CRP_active) and inactive (CRP_inactive) forms, but the major form is the inactive one (CRP_inactive) in the absence of cAMP. The role of cAMP binding is to shift the equilibrium toward the active form ([cAMP-CRP_active] ≫ [cAMP-CRP_inactive] and [cAMP₂-CRP_active] ≫ [cAMP₂-CRP_inactive]). The ligand cAMP can bind both to CRP_active and CRP_inactive, with the intrinsic affinities of k and k_i, respectively. The term K_c ([CRP_active]/[CRP_inactive]) is the population ratio of CRP_active versus CRP_inactive. The length of arrows indicates either the protein equilibrium or the strength of ligand affinity in a qualitative way. The scheme in panel A is shown for cAMP, but it can be generalized for other ligands such as adenosine. (B) Modified equilibrium-shift model for cGMP. The cGMP is hypothesized to bind to two different states (A-mode and B-mode) of CRP that are equilibrium linked through CRP_inactive, and one cGMP binding mode excludes the other. The A-mode binding of cGMP to CRP is hypothesized to stabilize the active form, similar to the simple equilibrium-shift model described in panel A. The B-mode binding of cGMP to CRP is hypothesized to stabilize the inactive form. In this scheme, the length of the arrows indicates our assumptions about protein equilibrium and cGMP binding affinity, but there is little experimental evidence.

FIG. 3.

Activation of WT CRP and CRP* variants by various concentrations of ligands. The DNA affinities of wild-type CRP (A), D53S CRP (B), and D53S/R82G CRP (C) were measured in various concentrations of the ligands cAMP (○), cGMP (⋄), and adenosine (▵). The in vitro DNA binding was measured by using a fluorescence anisotropy method. The concentration of each protein used was 100 nM. The scales of x axis (ligand concentration) and y axis (anisotropy value) are the same in the three panels. The solid lines in each panel indicate the best fits for the isotherms using the scheme in Fig. 2A and equations detailed in Materials and Methods.

FIG. 4.

Proteolytic digestion of WT CRP and CRP* variants by subtilisin. Each CRP (1.12 mg/ml) was digested with various amounts of subtilisin; the black wedge indicates twofold serial dilution of subtilisin, and the first lane contained 11.2 μg of subtilisin/ml (CRP/subtilisin, 1:100). An asterisk indicates the control lane showing each CRP treated identically but without subtilisin. Arrows indicate the uncut CRP band. The concentration of cAMP used for WT CRP was 100 μM. The reaction was carried out at 25°C for 24 h.