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. 2020 Feb 4;59(4):460-470.
doi: 10.1021/acs.biochem.9b00895. Epub 2020 Jan 10.

Structural Energy Landscapes and Plasticity of the Microstates of Apo Escherichia coli cAMP Receptor Protein

Rati Chkheidze, Wilfredo Evangelista  1 , Mark A White, Y Whitney Yin, J Ching Lee
Affiliations

Structural Energy Landscapes and Plasticity of the Microstates of Apo Escherichia coli cAMP Receptor Protein

Rati Chkheidze et al. Biochemistry. .

Abstract

The theory for allostery has evolved to a modern energy landscape ensemble theory, the major feature of which is the existence of multiple microstates in equilibrium. The properties of microstates are not well defined due to their transient nature. Characterization of apo protein microstates is important because the specific complex of the ligand-bound microstate defines the biological function. The information needed to link biological function and structure is a quantitative correlation of the energy landscapes between the apo and holo protein states. We employed the Escherichia coli cAMP receptor protein (CRP) system to test the features embedded in the ensemble theory because multiple crystalline apo and holo structures are available. Small angle X-ray scattering data eliminated one of the three apo states but not the other two. We defined the underlying energy landscape differences among the apo microstates by employing the computation algorithm COREX/BEST. The same connectivity patterns among residues in apo CRP are retained upon binding of cAMP. The microstates of apo CRP differ from one another by minor structural perturbations, resulting in changes in the energy landscapes of the various domains of CRP. Using the differences in energy landscapes among these apo states, we computed the cAMP binding energetics that were compared with solution biophysical results. Only one of the three apo microstates yielded data consistent with the solution data. The relative magnitude of changes in energy landscapes embedded in various apo microstates apparently defines the ultimate outcome of the cooperativity of binding.

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Figures

Figure 1.
Figure 1.
Cartoon showing the energy diagram of the three apo CRP microstates, as represented by different shapes and colors. Ki is the apparent binding constant for formation of the cAMP–CRP complex that subsequently binds to different promoter DNA sequences that can bend to different degrees.
Figure 2.
Figure 2.
Molecular structures of apo CRP (Protein Data Bank entry 3HIF) and holo CRP (Protein Data Bank entry 1G6N). The color code and identity of subunits are as follows: blue, subunit A; red, subunit B. (A) 3HIF AB, (B) 3HIF CD, (C) 3HIF EF, and (D) 1G6N.
Figure 3.
Figure 3.
Scattering curves calculated from the molecular structures in Figure 2. (A and B) Kratky plots and (C and D) log Guinier plots for simulated scattering curves derived from crystallographic structures. Panels B and D are focused on q = 0.1–0.25 to highlight the differences. Solid lines are calculated from the X-ray crystal structures of apo CRP (red, AB dimer; green, CD dimer; blue, EF dimer) under crystallization conditions. The dashed brown line (·-·) is calculated from holo CRP. Rg values for the simulated scattering curves: X-ray apo AB, 24.3 ± 0.016 Å; X-ray apo CD, 24.49 ± 0.015 Å; X-ray apo EF, 24.2 ± 0.015 Å; holo CRP, 24.1 ± 0.02 Å.
Figure 4.
Figure 4.
Comparison of the experimental SAXS data from apo wild-type CRP. (A and B) Kratky plots and (C and D) log Guinier plots. Experimental scattering data from apo wild-type CRP (●). Only alternate data points are shown for the sake of clarity. Fittings of the dimer models: AB dimer (green), χ2 = 1.8; CD (blue), χ2 = 2.2; EF (orange), χ2 = 1.7 (solid line under buffer conditions resembling the crystallographic conditions).
Figure 5.
Figure 5.
Bead model of apo CRP. (A) DAMMIF bead model of apo CRP (NSD = 0.47) shown with an X-ray dimer AB fit (χ2 = 1.8; NSD = 0.95), shown in both front and side views. (B) Same with X-ray dimer CD (χ2 = 1.6; NSD = 0.95).
Figure 6.
Figure 6.
Rigid body modeling of the apo CRP homodimer in solution. (A) Comparison of the Kratky plots of simulated and experimental SAXS data from apo wild-type CRP. (B) Bead model of apo CRP.
Figure 7.
Figure 7.
Plot of ln K vs residue number of apo and holo CRP. Symbols and subunit identity: filled circles, subunit A; empty circles, subunit B. Identities of structures: (A) 3HIF AB, (B) 3HIF CD, (C) 3HIF EF, and (D) 1G6N holo CRP.
Figure 8.
Figure 8.
Change in structural stability from apo to holo. A–F represent subunits in microstates AB, CD, and EF, respectively. Red and green data are for the NBD and DBD, respectively, of subunits A–F.
Figure 9.
Figure 9.
(A and B) Connectivity map (RSC) of subunits A and B of microstates 1 and 2, respectively, of apo CRP. The false color scale indicates positive and negative connectivity between residues. Positive connectivity indicates the folding–unfolding reactions of these residues are synchronized at the same time scale whereas negative connectivity indicates asynchronization between these residues. (C and D) Connectivity map (RSC) of microstate 3 and holo subunits A and B of apo and holo CRP. The false color scale indicates positive (red) and negative (blue) connectivity between residues. Positive connectivity indicates the folding–unfolding reactions of these residues are synchronized at the same time scale whereas negative connectivity indicates asynchronization between these residues.
Figure 10.
Figure 10.
Connectivity cross points in (left) apo CRP (PDB entry 3HIF) and (right) holo CRP (PDB entry 1G6N). The arrows show the locations of residues among the common connectivity crossing points in the pathways of signal transmission.
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