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. 2015 Sep 4;290(36):22225-35.
doi: 10.1074/jbc.M115.669267. Epub 2015 Jul 16.

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Philip D Townsend  1 , Thomas L Rodgers  2 , Laura C Glover  3 , Heidi J Korhonen  4 , Shane A Richards  5 , Lucy J Colwell  6 , Ehmke Pohl  3 , Mark R Wilson  7 , David R W Hodgson  7 , Tom C B McLeish  8 , Martin J Cann  9
Affiliations

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Philip D Townsend et al. J Biol Chem. .

Abstract

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.

Keywords: allosteric regulation; biophysics; calorimetry; cyclic AMP (cAMP); nucleoside/nucleotide analogue; protein dynamic.

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Figures

FIGURE 1.
FIGURE 1.
The influence of CAP-cAMP contacts on allostery in CAP. a, ribbon diagram of the x-ray crystal structure of CAP (Protein Data Bank code 4HZF) showing the secondary and tertiary structures of the CAP homodimer with cAMP bound. The inset shows the hydrogen-bonding network at the cAMP binding site in the wild type protein. The structure and inset are shown in different orientations for clarity. The labeled amino acids in the inset contact cAMP and are analyzed in this study. b, B-factor plotted against amino acid number for the crystal structure and the manually curated ENM for chain A and chain B of the CAP crystal structure. c, the change in cooperativity (K2/K1) that occurs when kR/k is varied at the indicated residue. d, the change in cooperativity (K2/K1) that occurs when kR/k is varied for pairs of indicated residues.
FIGURE 2.
FIGURE 2.
Mapping local dynamics in CAP. The effect of mutation of Gly-71/Glu-72 (a), Arg-82/Ser-83 (b), and Thr-127/Ser-128 (c) on local dynamics over the CAP monomer. The chart represents the percentage variation in the calculated B-factor from the wild type curated ENM plotted against amino acid number. The ENMs represent the single ligand-bound state with one molecule of cAMP bound to chain A.
FIGURE 3.
FIGURE 3.
Disruption of CAP-ligand interactions through the use of cAMP analogs. a, the distribution of binding interactions between cAMP and wild type CAP. b–d, binding interactions between Sp-cAMPS (b), cIMP (c), and 2′-deoxy-cAMP (d) and CAP. e–g, ITC trace (upper panel) and binding isotherm (lower panel, the different colored symbols represent individual experiments) for the calorimetric titration of cAMP (e), Sp-cAMPS (f), and cIMP (g) to CAP are shown. The thermodynamic parameters obtained are shown in Table 2.
FIGURE 4.
FIGURE 4.
Analysis of CAP protein structure. a, overlay of the x-ray crystal structures of wild type CAP with cAMP (green) and wild type CAP with Sp-cAMPS (gold). b, close-up of the ligand-binding site of wild type CAP with cAMP showing the hydrogen-bonding network. c, close-up of the ligand-binding site of wild type CAP with Sp-cAMPS showing hydrogen-bonding network. d, overlay of ligand-binding sites of wild type CAP with cAMP and Sp-cAMPS.
FIGURE 5.
FIGURE 5.
The influence of CAP-cAMP contacts on changes to allostery induced by second site mutations. The charts show the change in allosteric cooperativity induced by mutations in ENMWT (ΔK2/K1 ENMWT) subtracted from the change in allosteric cooperativity induced by the same mutation in ENMG71/E72 (ΔK2/K1 ENMG71/E72) (a), ENMR82/S83 (ΔK2/K1 ENMR82/S83) (b), or ENMT127/S128 (ΔK2/K1 ENMT127/S128) (c) plotted against amino acid number in CAP. The colored backgrounds indicate the cAMP-binding domain (pale blue), interface forming α helix (white), and DNA-binding domain (red).
FIGURE 6.
FIGURE 6.
Correlations between allostery and motion in CAP. The y axes of all charts show the degree of correlated motion between a particular cAMP-contacting residue and every other amino acid in the CAP monomer. The x axes of all charts show the change in allosteric cooperativity induced by individual mutation of every amino acid in ENMWT (ΔK2/K1 ENMWT) subtracted from the change in allosteric cooperativity induced by the same mutation in the ENM with paired mutations in cAMP-contacting residues identified in Fig. 1D (ENM named as per Fig. 5). a, correlated motion between Gly-71 and all other amino acids plotted against ΔK2/K1 ENMWT subtracted from ΔK2/K1 ENMG71/E72. b, correlated motion between Glu-72 and all other amino acids plotted against ΔK2/K1 ENMWT subtracted from ΔK2/K1 ENMG71/E72. c, correlated motion between Arg-82 and all other amino acids plotted against ΔK2/K1 ENMWT subtracted from ΔK2/K1 ENMR82/S83. d, correlated motion between Ser-83 and all other amino acids plotted against ΔK2/K1 ENMWT subtracted from ΔK2/K1 ENMR82/S83. e, correlated motion between Thr-127 and all other amino acids plotted against ΔK2/K1 ENMWT subtracted from ΔK2/K1 ENMT127/S128. f, correlated motion between Ser-128 and all other amino acids plotted against ΔK2/K1 ENMWT subtracted from ΔK2/K1 ENMT127/S128. g, correlated motion between 35 covarying residue pairs with at least one cAMP-contacting residue from an alignment of CAP variants plotted against ΔK2/K1 ENMWT subtracted from ΔK2/K1 for the appropriate ENM for that cAMP-contacting residue (ΔK2/K1 ENMALL). All analysis has been performed for chain A of the fully cAMP bound ENM.
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