Figure 1.
Phosphorylation Can Perturb the Energy Landscape of a Protein to Cause Changes in Conformation and Dynamics
This figure is a visual representation of such changes. In this work, we predict the structural change of proteins due to phosphorylation by locating the new global energy minimum of the energy surface.
Figure 2.
Example of the Hierarchical Loop Prediction, Applied to Reconstructing the Phosphorylated Activation Loop of CDK2/Cyclin A
In this example, only the loop is predicted, and the remainder of the protein is held rigid in its crystallographic conformation.
(A) Backbone traces for the ten lowest-energy loops sampled in the initial build-up stage (left) and the third and final refinement stage (right) of the hierarchical loop prediction protocol (figures prepared with Chimera [72]).
(B) Energies as a function of backbone RMSD values of the 20 lowest-energy loops sampled in the four stages of the loop prediction protocol.
(C) Energies as a function of the error in the phosphorus atom position, relative to the crystal structure, for the 20 lowest-energy loops sampled in the four stages of the loop prediction protocol.
Figure 3.
Loop Reconstruction and Prediction in CDK2/Cyclin A
(A) Reconstruction: Crystal structure of phosphorylated CDK2/cyclin A (blue) and the predicted loop structure (red). The starting structure for the prediction was the phosphorylated structure, with the only difference being the loop region.
(B) Prediction: Crystal structure of phosphorylated CDK2/cyclin A (blue), the crystal structure of the unphosphorylated CDK2/cyclin A (green), and the predicted loop structure upon in silico phosphorylation of the unphosphorylated CDK2/cyclin A structure (red). Figures prepared with Chimera [72].
Table 1.
Results from the CDK2 Case Study
Figure 4.
Detailed View of One Portion of the Structural Superposition between the Phosphorylated and Unphosphorylated Crystal Structures of CDK2/Cyclin A
The phosphorylated activation loop (blue) passes through the middle of Tyr179 (CPK) when inserted into the non-phosphorylated structure (green) of CDK2. Figure prepared with Chimera [72].
Figure 5.
The 20 lowest-energy loops predicted for (A) reconstruction of residues 152–163 in phosphorylated, cyclin-bound CDK2; and (B) prediction of the structure of residues 152–163 upon in silico phosphorylation of Thr160 in the unphosphorylated, cyclin-bound CDK2. In each case, the loop and surrounding side chains are optimized simultaneously as described in Materials and Methods.
Figure 6.
Differences in Conformational Predictions for Phosphorylated CDK2 with and without Cyclin A Bound
The 20 lowest-energy loops are considered for predicting the conformation of residues 152–163 upon in silico phosphorylation of Thr160 (A) in cyclin-bound CDK2 and (B) CDK2 in the absence of cyclin A. In (B), the x-axis represents the deviation of the phosphate from its position in the fully activated CDK2/cyclin A complex; the activation loop and pThr160 are disordered in the crystal structure of phosphorylated CDK2 in the absence of cyclin A. These calculations were performed with a consistent set of parameters that do not bias the results toward well-ordered loop structures. In the absence of cyclin A, the phosphate does not localize to the Arg cluster as in the cyclin bound case, and the 20 lowest-energy structures show considerable diversity in conformation.
Figure 7.
Active and Inactive Conformations of the CDK2 Activation Loop
Left: Blue represents the crystal structures of the phospho-CDK2/cyclin A complex, and green represents the T160E-predicted active-like conformation. The Arg cluster is shown in stick representation. The carboxylate group of Glu160 and the phosphate group of pThr160 are almost exactly superimposed. Right: Purple represents the crystal structure of unphosphorylated CDK2/cyclin A, and yellow represents the predicted inactive conformation of T160E. These two structures are qualitatively similar in that Thr160 and Glu160 both point out into solvent and the Arg cluster is better solvated. Figures prepared with Chimera [72].
Table 2.
Energy Differences (kcal/mol) between “Active” and “Inactive” Conformations of the Activation Loop
Table 3.
Proteins Used in Our Test Set
Table 4.
Reconstruction of Phosphorylated Loop Conformations
Table 5.
Prediction of Phosphorylated Loop Conformations
Figure 8.
Helix Reconstruction and Prediction of FixJ
The loop-helix-loop region of FixJ was predicted starting from either the phosphorylated structure (blue) or the unphosphorylated structure (unpublished results). The reconstruction (starting from the phosphorylated structure) is in red, and the prediction (starting from the unphosphorylated structure) is in green. Figure prepared with Chimera [72].