Differential modulation of energy landscapes of cyclic AMP receptor protein (CRP) as a regulatory mechanism for class II CRP-dependent promoters

Abstract

The Escherichia coli cAMP receptor protein, CRP, is a homodimeric global transcription activator that employs multiple mechanisms to modulate the expression of hundreds of genes. These mechanisms require different interfacial interactions among CRP, RNA, and DNA of varying sequences. The involvement of such a multiplicity of interfaces requires a tight control to ensure the desired phenotype. CRP-dependent promoters can be grouped into three classes. For decades scientists in the field have been puzzled over the differences in mechanisms between class I and II promoters. Using a new crystal structure, IR spectroscopy, and computational analysis, we defined the energy landscapes of WT and 14 mutated CRPs to determine how a homodimeric protein can distinguish nonpalindromic DNA sequences and facilitate communication between residues located in three different activation regions (AR) in CRP that are ∼30 Å apart. We showed that each mutation imparts differential effects on stability among the subunits and domains in CRP. Consequently, the energetic landscapes of subunits and domains are different, and CRP is asymmetric. Hence, the same mutation can exert different effects on ARs in class I or II promoters. The effect of a mutation is transmitted through a network by long-distance communication not necessarily relying on physical contacts between adjacent residues. The mechanism is simply the sum of the consequences of modulating the synchrony of dynamic motions of residues at a distance, leading to differential effects on ARs in different subunits. The computational analysis is applicable to any system and potentially with predictive capability.

Keywords: RNA polymerase; allosteric regulation; bacterial signal transduction; bacterial transcription; energy landscapes; long-range communication; signaling pathway; thermodynamics; transcription factor; transcription regulation.

Figure 1.

Models of the structural architectures of class II and class I CRP-dependent promoters. The dark and light green subunits of CRP represent subunits A and B, respectively. The blue structure represents α-CTD of RNAP. The black line is the DNA molecule, and the gray box in the DNA represents the –TGTGA– half-site.

Figure 2.

A, structure of subunit A of holo WT CRP showing the spatial locations of ARs, key residues, and the distances between them. B, crystallographic structure of holo WT CRP (PDB code 1I5Z) at pH 7.5 and 1.9 Å resolution. Subunits A and B are shown in green and magenta, respectively. NBD and DBD denote the cAMP- and DNA-binding domain, respectively.

Figure 3.

A, curve-fitted and inverted second-derivative amide I spectrum of apo-CRP. The inversion of the second-derivative spectrum was done by factoring by −1. The parameters for curve fitting are summarized in Table 2. B, amide I spectra as a function of time of apo- and cAMP-liganded H159L mutant in a 50:50 H₂O/D₂O. The spectra were recorded at 1, 10, 20, 30, 40, and 60 min (starting times) after the sample mixing. The spectrum of WT apo-CRP in H₂O, as shown as a black short broken line, is included for reference. The arrows indicate the direction of change in absorbance as a function of time. C, amide I spectra as a function of time of apo- and cAMP-liganded K52N CRP mutant in a 50:50 H₂O/D₂O. The spectrum of WT apo-CRP in H₂O, as shown as a black short broken line, is included for reference. The arrows indicate the direction of change in absorbance as a function of time. D, amide I spectra as a function of time of apo- and cAMP-liganded K52N/H159L CRP mutant in a 50:50 H₂O/D₂O. The spectrum of WT apo-CRP in H₂O, as shown as black short broken line, is included for reference. The arrows indicate the direction of change in absorbance as a function of time.

Figure 4.

Relative intensity changes of the β-sheet band (∼1634 cm⁻¹) (A) and the α-helix band (1656–1653 cm⁻¹) (B) of three mutants and WT CRP in a 50:50 H₂O/D₂O solution as functions of time. The values of relative intensities were normalized. The samples and symbols are as follows: WT (1); D159L (2); K52N/D159L (3); and K52N (4). Data points in red and black are for holo and apo-CRP, respectively. The error for the short time points (1–5 min) is ±2% for the β-sheet band but is ±5% for the α-helix band.

Figure 5.

Residue stability of holo CRP residues for the A and B subunits, respectively. Thick black line and thick red or blue lines are residues for WT and K52N mutant, respectively. Numbers in boxes show the residues.

Figure 6.

〈lnK_{i, f}〉 of CRP subunits and domains as a function of mutations. Odd- and even-numbered samples represent those of subunits A and B, respectively. The color codes are red and blue for subunits A and B, respectively; ○ and ▵ for NBD and DBD, respectively. The order of mutant presented is as follows: 1–2 (WT); 3–4 (H19L); 5–6 (H21L); 7–8 (K52N); 9–10 (H58A); 11–12 (H58K); 13–14 (E96A); 15–16 (K101D); 17–18 (T158A); 19–20 (H159L); 21–22 (K52N-H159L); 23–24 (52–159-101); 25–26 (T58K + 23/24); 27–28 (T58A + 23/24); 29–30 (E181V).

Figure 7.

lnK of AR₁ residues in subunits A and B as a function of mutations. The identities of mutations are the same as in Fig. 6. The color codes for residues are as follows: red circle, 62; black circle, 66; green circle, 148; blue circle, 152; pink circle, 156; light green circle, 158; and gray circle, 163.

Figure 8.

lnK of AR₂ residues in subunits A and B as a function of mutations. The identities of mutations are the same as in Fig. 6. The color codes for residues are as follows: subunit A: black circle, 21; red circle, 37; green circle, 98; subunit B: blue triangle, 21; pink triangle, 37; cyan triangle, 98.

Figure 9.

lnK of AR₃ residues in subunits A and B as a function of mutations. The identities of mutations are the same as in Fig. 6. The color codes for residues are as follows: subunit A: black circle, 19, 21, 32, and 34; black triangle, 52, 54, and 55; subunit B: red circle, 19; green circle, 21; blue circle, 32; *, 34; black triangle, 52, 54, and 55.

Figure 10.

A and B are connectivity map (RSC) of WT and K52N subunits A and B, respectively. The false color scale indicates positive and negative connectivity between residues. Positive connectivity indicates the folding–unfolding reactions of these residues are synchronizes at the same time scale, whereas negative connectivity indicates asynchronization between these residues.