Conformational frustration in calmodulin-target recognition

Abstract

Calmodulin (CaM) is a primary calcium (Ca(2+) )-signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM-binding domains from two Ca(2+) -CaM-dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM-CaMKII complex than in the CaM-CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C-domain of CaM that need to be broken before the formation of intermolecular contacts with the N-domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations.

Keywords: binding frustration; binding route analysis; calmodulin; calmodulin-binding targets; coarse-grained molecular simulations; protein-protein association; target recognition.

Figure 1

Native structures of the CaM-CaMBT complexes (A) and (B) show the PDB structures of the CaM-CaMKI (PDB ID: 2L7L) (Gifford et al. 2011) and CaM-CaMKII (PDB ID: 1CDM) (Meador et al. 1993) complexes. Helices and the Ca²⁺-binding loops of CaM are denoted by A-H and I-IV, respectively in (A) and (B). CaM is colored red (N-terminal domain) and blue (C-terminal domain) and the Ca²⁺ ions are represented with yellow spheres. The CaMKI and CaMKII peptides are shown in green. The sequence of CaMKI and CaMKII are indicated below (A) and (B), respectively with one letter code. The hydrophobic residues of the CaMBTs from the hydrophobic motifs (1-5-10 and 1-14 for CaMKI and 1-5-10 for CaMKII) in both the sequences are colored in red.

Figure 2

Interactions between the CaM domains and the CaMBTs during association. (A), (B) and (C) represent three successful trajectories for the association of CaM-CaMKI projected on the 2D distribution (in log-scale) –ln P(Z_n, Z_c) of all the trajectories. Similarly, (D), (E) and (F) are for CaM-CaMKII. The trajectories are colored by a normalized time from blue to red. The 2D distribution is colored from black to white proportional to the population (from high to low). See the Models and Method section for the definition of Z_n and Z_c. The basins (I and II in A–C for CaM-CaMKI, and I′ and II′ in D–F for CaM-CaMKII) are indicated by the black arrows.

Figure 3

The binding route analysis on the trajectories from the successful associations between CaM and the CaMBTs. (A) and (B) illustrate the formation of the normalized intermolecular contacts $\overline{Z_n}$ and $\overline{Z_c}$ from the N- and C-terminal domain of CaM compared to the average Z̄ (normalized) and the targets CaMKI and CaMKII, respectively. For both systems Z̄ is defined as the total number of intermolecular contacts between CaM and the target, such that $\overline{Z}=\overline{Z_n+Z_c}$. See the Models and Method section for a detailed discussion on Z̄, $\overline{Z_n}$ and $\overline{Z_c}$. In each plot, the diagonal line is drawn as a reference for the intermolecular contacts $\overline{Z_n}$ or $\overline{Z_c}$ that ideally follow the average Z̄. The vertical dashed lines in the plots indicate the values of Z̄ for which the corresponding values of $\overline{Z_n}$ and $\overline{Z_c}$ differ significantly during the association for both systems.

Figure 4

Probability of contact formation between CaM and CaMKI along the binding route. (A), (B) and (C) represent the contact maps calculated between the amino acids from the side-chain of CaM and CaMKI, at the different values of Z̄ ~0.3, 0.6 and 0.8 (as indicated in Fig. 3 (A)), respectively. A linearized model of CaM is shown below the plots with the individual helices displayed as cylinders, the linkers connecting the helices shown as lines, and the N-terminal and C-terminal helices are shown in red and blue, respectively.

Figure 5

Probability of contact formation between CaM and CaMKII along the binding route. (A), (B) and (C) represent the contact maps calculated between the amino acids from the side-chain of CaM and CaMKII, at the different values of Z̄ ~0.2, 0.3 and 0.6 (as indicated in Fig. 3(B)), respectively. A linearized model of CaM is shown below the plots with the individual helices displayed as cylinders, the linkers connecting the helices shown as lines, and the N-terminal and C-terminal helices are shown in red and blue, respectively.

Figure 6

Conformational frustration in CaM-CaMBT recognition. (A), (B) and (C) represent the schematic diagram of CaM-CaMBT association process along the binding route (see Fig. 3). The unstructured CaMBT (CaMKI or CaMKII) initially interacts with one of the domain of CaM (predominantly the cCaM). At the early stage of association (from A to B) the conformational frustration comes into play as the CaMBT starts to interact with the other domain of CaM (predominantly the nCaM). The conformational frustration at the late stage (from B to C) occurs, as the CaMBT approaches towards the binding pocket of CaM. As a result, intermolecular contacts between CaM and CaMBT are either broken or new intermolecular contacts are formed. In the schematic diagram of CaM the number 1-4 denotes the four EF-hands. The BC and FG helix-linkers of CaM are represented by the dotted lines in red and blue, respectively. Yellow spheres denote positions of four Ca²⁺ ions. The binding loops of CaM are not shown. The ensemble of CaMBT structures is shown in green.

Figure 7

Difference in the intermolecular contact probability of the residues from the CaMBTs along the binding route at the early stage of association. (A) Difference in the contact probability (ΔP_i) for each residue of CaMKI, at Z̄ ~0.3 and Z̄ ~0.6 (see Fig. 3A in the Main text). (B) ΔP_i for each residue of CaMKII, at Z̄ ~0.2 and Z̄ ~0.3 (see Fig. 3B in the Main text). The sequences of CaMBTs are shown on top of each plot. The first hydrophobic residues from the binding motif of the CaMBTs are shown in green (W303 from CaMKI and L299 from CaMKII).