A C-helix Residue, Arg-123, Has Important Roles in Both the Active and Inactive Forms of the cAMP Receptor Protein

Abstract

The cAMP receptor protein (CRP) of Escherichia coli exists in an equilibrium between active and inactive forms, and the effector, cAMP, shifts that equilibrium to the active form, thereby allowing DNA binding. For this equilibrium shift, a C-helix repositioning around the C-helix residues Thr-127 and Ser-128 has been reported as a critical local event along with proper ␤4/␤5 positioning. Here we show that another C-helix residue, Arg-123, has a unique role in cAMP-dependent CRP activation in two different ways. First, Arg-123 is important for proper cAMP affinity, although it is not critical for the conformational change with saturating amounts of cAMP. Second, Arg-123 is optimal for stabilizing the inactive conformation of CRP when cAMP is absent, thereby allowing a maximal range of regulation by cAMP. However, Arg-123 does not appear to be critical for a functional response to cAMP, as has been proposed previously The cAMP receptor protein (CRP)2 of Escherichia coli is one of the best-studied transcriptional activators (1-3). It is a homodimeric protein that binds specific DNA sequences with high affinity in response to binding a molecule of cAMP to each monomer. In cases where it activates gene expression, the DNA-bound CRP interacts with RNA polymerase to stabilize polymerase binding and, thus, stimulate transcription.CRP is a classic allosteric protein, each subunit of which is composed of two distinct domains connected by a hinge region; this is, an N-terminal effector binding domain and a C-terminal DNA binding domain (4). cAMP binding to the effector binding domain triggers a conformational change in the protein that leads to a precise repositioning of the DNA binding domains that promotes binding to its target DNA sequence. CRP exists in equilibrium between an active form that can bind specific DNA target sequences and an inactive form that cannot. In this view, cAMP binding to the protein shifts the equilibrium toward the active form either by destabilizing the inactive form or by stabilizing the active form.Although an enormous amount is known about the behavior of the DNA binding form of CRP, relatively less is known about the process of activation by cAMP binding. The critical obstacle has been the absence of a crystal structure of the cAMP-free form, although several structures of active CRP are known both in the presence and absence of DNA (4 -6). A number of research groups have used other methods to better understand the cAMP-dependent activation of CRP protein (3). We have also contributed to this field by identifying two distinctive local effects of cAMP binding in the activation process (7); (i) C-helix repositioning through direct interaction of cAMP with two C-helix residues Thr-127 and Ser-128 and (ii) the concomitant reorientation of the ␤4/␤5 loop.Recently another group compared the structures of a large number of cAMP-binding proteins of quite diverse function (8). They showed that despite large functional differences there were underlying structural similarities in both cAMP sensing a...

Two-State Allosteric Modeling Suggests Protein Equilibrium as an Integral Component for Cyclic AMP (cAMP) Specificity in the cAMP Receptor Protein ofEscherichia coli

“…As suggested previously (24,30,40), CRP also exists in an equilibrium between active and inactive forms even in the absence of cAMP, and the role of cAMP binding to the protein is to shift the equilibrium toward the active form. Based on this well-known concept, we have applied an equilibrium-shift model that quantitatively connects the equilibrium poise with the ligand specificity in the CRP* variants, as well as WT CRP.…”

“…CRP is a homodimer in which each subunit contains two domains, the cAMP-binding domain and the DNA-binding domain, separated by a hinge region (27). The structure of the inactive form of CRP has never been determined, so the mechanism of allosteric activation by cAMP is conjectural, but plausible models for the conformational change that cAMP effects have been proposed (29,39).CRP variants have been found that have altered ligand specificity (1,5,9,14,19,23,40). The substitutions in some of these variants lie in the effector-binding pocket itself and presumably alter the specific interactions between CRP and the ligand (23, 40).…”

“…CRP variants have been found that have altered ligand specificity (1,5,9,14,19,23,40). The substitutions in some of these variants lie in the effector-binding pocket itself and presumably alter the specific interactions between CRP and the ligand (23, 40).…”

“…The substitutions in some of these variants lie in the effector-binding pocket itself and presumably alter the specific interactions between CRP and the ligand (23,40). Other CRP variants are fundamentally different and more difficult to explain.…”

“…The detailed procedure is described elsewhere (40). The binding isotherms were analyzed using equation 4 with an assumption that the anisotropy signals are the same for all P a L n D regardless of the number of ligand bound (n).…”