Figure 1.
Holo CRP crystal structure and functional sites.
A. The CRP structure in complex with DNA and cAMP (PDB: 1CGP). The N-terminal cAMP binding domains (V1-N133), the C-terminal DNA binding domains (V139-R209) and the hinge regions (L134-D138) connecting the two domains in subunits A/B are represented in cyan/ice blue, dark pink/light pink and green/yellow, respectively. The α-helices C, D, E, F and bound cAMP molecules are indicated for both subunits. B. The residues for the primary cAMP binding -G71, E72, R82, S83, T127, S128 (subunit B)-, the secondary cAMP binding –E58, A135, R180-, the specific DNA binding –R180, E181, R185-, the nonspecific DNA interactions –R169, Q170, S179, and the RNAp interaction –A156-Q164 (AR1) are labeled. The residues are colored white, blue, red and green corresponding to the residue types non-polar, basic, acidic and polar, respectively. The approximate position of the secondary cAMP is placed, considering the dimer CRP with four cAMPs (PDB: 2CGP). Figures are generated using VMD 1.8.7 [68].
Figure 2.
Dynamic fluctuations of apo CRP monomer/dimer and holo CRP by MD simulations.
A. RMSD of the sampled conformations from the initial energy-minimized structure for apo CRP monomer/dimer and holo CRP along the 150 ns MD simulations. Comparisons between the RMSDs of different states are given in the inset figure. B. The MSF of residues for apo CRP monomer/dimer and holo CRP. The values are averaged over parallel runs for all states, and averaged over subunits for the dimers. The difference-MSF of different states is given as a sub-plot. C. The difference-MSF of apo CRP dimer (averaged over subunits) and apo CRP monomer.
Figure 3.
Dynamics network of apo CRP monomer/dimer and holo CRP by MD simulations.
The correlation between residue fluctuations based on first ten essential modes is calculated as an average over three parallel MD runs for apo CRP monomer/dimer and holo CRP MD trajectories. The correlation maps are given for: (A) apo CRP monomer AVG, (B) apo CRP dimer AVG, and (C) holo CRP (single run).
Figure 4.
GNM cross-correlations of apo/holo CRP and the switch behavior.
Comparison of the correlations between residue fluctuations in average ten slowest modes for the apo CRP NMR solution structure (PDB: 2WC2, model 1) and holo CRP X-ray crystal structure with the two cAMPs bound (PDB: 1G6N). Below, the ribbon diagrams color coded with the correlation values of the L134-D138 hinge (average) with the rest of the structure are given. The difference-correlation map of the two correlation maps is given in the middle.
Figure 5.
The alignment of MD cluster best members of apo CRP monomer/dimer and holo CRP.
The superimposed MD best member structures of the clusters generated from three apo CRP monomer runs (A, B, C), three apo CRP dimer runs (D, E, F) and a holo CRP run (G) with a cluster radius of 3.5 Å. Colors of the structures match the colors of the clusters given in RMSD plots of cluster evolution in time (Figure S3). For example, the structure in cyan is the cluster best member no. 3, which is also colored in cyan. The white structures are the starting conformations for each run.
Figure 6.
Ensemble of CRP conformations.
Ensemble of structures (dynamic states) are selected according to their dynamic behavior among the MD cluster best member structures with a cluster radius of 3.5 Å. The ribbon diagrams are color coded with the correlation values of the L134-D138 region (average) with the rest of the structure. The difference-correlations maps for each structure in the ensemble with respect to the holo CRP dimer are given: (A) The L134-D138 hinge of both subunits (A/a& B/b) displays apo-like behavior with relatively strong coupling with the DNA binding domain of its own subunit (higher positive values), relatively weak coupling with the L134-D138, K52-E58 and G173-V176 regions of the other subunit (higher negative values), and weak coupling between the K52-E58 and G173-V176 sites (higher negative values). (B) This structure is the one that displays the closest dynamic behavior in overall to the holo state with respect the both subunits (A/a&B/b); theL134-D138 hinge is not strongly correlated with the DNA binding domain of its own subunit (lower positive values), and not weakly correlated with the K52-E58 and G173-V176 sites (lower negative values) compared to (A). (C) Subunit A/a displays more holo-like behavior while subunit B/b assumes apo-like behavior following the L134-D138 hinge dynamics. See (B) for the dynamics of subunit A/a and see (A) for the dynamics of subunit B/b.
Table 1.
Details of the simulated systems.
Table 2.
Cluster populations.