Fig 1.
Overview of the CAP structure and allostery.
(A) CAP homo-dimer in the two-liganded state known from X-ray diffraction (PDB id: 1G6N [9]). The first protomer is depicted in white cartoon and the second is in yellow cartoon, the two cAMP are represented as balls. (B) Representation of the important region of CAP, the DNA Binding Domain (DBD) is in green, the Nucleotide Binding Domain (NBD) in blue and hot-spots delineated by FDA are in red, the cAMP is in grey balls. (C) Schematic representation of cAMP binding to CAP and allosteric communications between the two protomers. The DBD is shown in green, the NBD is shown in blue. The first cAMP (orange square) binds with a high affinity, whereas the second cAMP binds with a lower affinity (negative cooperativity). The cAMP binding provokes conformational changes in the DBD allowing DNA binding.
Fig 2.
(A) Estimated entropic contributions-TΔS to the binding free energy of the first (red) or the second (blue) cAMP binding event, and the overall entropy change for the binding of both cAMP (purple). Estimates from force covariance (FC) and quasi-harmonic (QH) analyses of either protein main chain (“MC”) or the full protein including DBD (“full”). For comparison, NMR-based estimates (“NMR”) are given for the entropy change of the first and second binding event of a truncated CAP construct (“CBD”) without DBD [8] and, respectively, for the binding of both cAMP to the full protein (“full”) [23]. (B) Functional motion of CAP for DNA binding as sampled in MD simulations. Projection of CAP X-ray structures and all simulation data from apo (black), cap1 (blue) and cap2 (orange) states on the first eigenvector obtained from a PCA of available 2 cAMP-bound X-ray structures, either solved in absence of DNA (1–6: 1GN6 [9], 1HW5 [27], and 1I6X, 3RDI, 3ROU, 1I5Z –all unpublished) or in presence of DNA (9–11: 1RUO [28], 1RUN [28] and 1CGP [26]). The two intermediate structures (PDB ids: 3QOP (unpublished) and 3KCC [29]) are not bound to DNA but to two cAMP molecules, localized in between the DBD and β-strand 5, triggering a rotation of the DBD.
Fig 3.
Allosteric network upon binding the first cAMP obtained from FDA.
Residue pairwise forces difference between the apo and the cap1 states are shown as blue sticks at a 50 pN (A) and 40 pN cut-off (B) for the CAP homo-dimer represented as cartoon. The first and second protomers are depicted in white and yellow, respectively. The cAMP molecule (white) and key residues are represented as sticks. (C, D) Zoom of (B) to highlight two distinct allosteric connection pathways termed A (C) and B (D) in the allosteric network between the two protomers at a cut-off value of 40 pN. Note that D is rotated by 180 degrees with respect to C.
Fig 4.
Conformational ensemble of Leu73 (A) and Arg123’ (B), involved in pathway A, for the apo (black), the cap1 (blue) and the cap2 (orange) states.
The protein is represented as cartoon in white (first protomer) and yellow (second protomer). Key residues are represented as cyan sticks. Histograms of local RMSDs are shown. The average structure of all CAP simulations was used as reference structure for the fitting and RMSD calculation, allowing to directly compare conformations of different states. Only residues around a 6Å cut-off were used for the fitting to track local rearrangements without including global motions.
Fig 5.
Allosteric network upon binding the second cAMP obtained from FDA.
Residue pairwise force differences between the cap1 and cap2 states are shown as blue sticks at (A) 50 pN and (B) 40 pN cut-off. (C) Zoom of B highlighting the allosteric connection pathway between the two protomers, which resembles pathway B in Fig 3D. (D) Zoom of B, highlighting the force changes in the Glu58-Arg87 and Glu58-Q174 pairs which connects the NBD with the DBD and which are symmetrically present in both protomers.
Fig 6.
Glu58 and Arg87 minimal distance decreases upon activation.
Probability distribution of the minimal distance between Glu68 and Arg87 for apo (black), cap1 (blue) and cap2 (orange) states. Both protomers were taken into account. Vertical dashed lines represent the respective values as observed in the cap2 X-ray structure (1G6N) and the range covered in the apo NMR structures (2WC2 [25]). The integration limits used in the inset for the three states (activated, intermediate and deactivated) have been determined by the two inflection points of the cap2 distribution (2.71 and 5.11 for intermediate and deactivated respectively).