Identifying allosteric fluctuation transitions between different protein conformational states as applied to Cyclin Dependent Kinase 2

Abstract

Background: The mechanisms underlying protein function and associated conformational change are dominated by a series of local entropy fluctuations affecting the global structure yet are mediated by only a few key residues. Transitional Dynamic Analysis (TDA) is a new method to detect these changes in local protein flexibility between different conformations arising from, for example, ligand binding. Additionally, Positional Impact Vertex for Entropy Transfer (PIVET) uses TDA to identify important residue contact changes that have a large impact on global fluctuation. We demonstrate the utility of these methods for Cyclin-dependent kinase 2 (CDK2), a system with crystal structures of this protein in multiple functionally relevant conformations and experimental data revealing the importance of local fluctuation changes for protein function.

Results: TDA and PIVET successfully identified select residues that are responsible for conformation specific regional fluctuation in the activation cycle of Cyclin Dependent Kinase 2 (CDK2). The detected local changes in protein flexibility have been experimentally confirmed to be essential for the regulation and function of the kinase. The methodologies also highlighted possible errors in previous molecular dynamic simulations that need to be resolved in order to understand this key player in cell cycle regulation. Finally, the use of entropy compensation as a possible allosteric mechanism for protein function is reported for CDK2.

Conclusion: The methodologies embodied in TDA and PIVET provide a quick approach to identify local fluctuation change important for protein function and residue contacts that contributes to these changes. Further, these approaches can be used to check for possible errors in protein dynamic simulations and have the potential to facilitate a better understanding of the contribution of entropy to protein allostery and function.

Figure 1

Comparison of GNM and B Factor Profiles for CDK2. (A) Structural mapping of GNM dynamic modeling results for the apo form of CDK2 (PDB ID: 1 HCL; see Materials and Methods). The N-terminal lobe (top) is more flexible (red) when compared to the C-terminal lobe (blue). (B) Comparison of modal plots for the two largest amplitude fluctuations are shown for the apo (red) and ATP bound (blue) conformers of ATP. Flexible regions defined by the GNM differs from those defined by experimental temperature factors (dashed line). Locating bars at top represent (a) N-terminal lobe, (b) PSTAIRE helix, (c) T-Loop, and (d) G-loop.

Figure 2

TDA on the CDK2 Activation Cycle. Fluctuation changes detected by TDA_mode are mapped to individual structures of the CDK2 activation cycle. To fully activate the kinase, a series of steps must occur involving (1) ATP binding, (2) cyclin A binding (cyclin not shown), and (3) T160 phosphorylation leading to (4) substrate binding (substrate not shown). Values have been normalized such that positive and negative values respectively indicate increasing (red) and decreasing (blue) fluctuations when compared to the previous conformational state. ATP is shown in yellow.

Figure 3

Fluctuation and Structural Changes Detected in the Activation Cycle of CDK2. Deviation of fluctuation changes (Z_diff) between different CDK2 conformers: (A) apo and ATP bound structure, (B) ATP bound and cyclin binding, (C) phosphorylated and unphosphorylated T160, and (D) fully activated CDK2 and substrate bound conformer. Structural and fluctuation changes are plotted separately against the residue index of CDK2 only. Different fluctuation changes are observed for the kinase when comparing large amplitude modes (solid lines) and experimental temperature factors (dashed lines) between conformers. Significant changes are identified based on the threshold of 1.5 standard deviations from the mean fluctuation centered at zero (red lines). Structural changes are measured by RMSD between Cα atoms. At top, black bars mark regions of particular interest: (a) N-terminal lobe, (b) PSTAIRE Helix, (c) T-Loop, (d) G-Loop, (e) residues 34–47 important for phosphoryl transfer as well as substrate binding, and finally (f) the CDK insert that is an important binding site for other regulatory proteins.

Figure 4

Entropy Compensation in CDK2. An example of entropy compensation in CDK2 serving as a possible mechanism for protein allostery. The transitional changes from a semi-active to active state (dashed line) and finally from an active state to substrate bound state (solid line) are shown. The changes in fluctuation between the (a) G-loop and (b) T-loop are observed to have an inverse relationship with each other.

Figure 5

PIVET Results Between Apoenzyme and ATP Bound CDK2 Conformer. Residue pair changes between apo and ATP bound conformers of CDK2 and their effect on global fluctuation. 27 residue pairs were observed to have a change in neighboring. Shown are: gain of a neighbor (red), loss of a neighbor (blue), loss of 2 neighbors (green), and no net change observed (orange). The table lists the impact of residue pairs as a percent of overall structure and direction of change where -1 indicate loss of a neighbor (disruption) and 1 a gain of a neighbor in going from the apo to the ATP bound conformer.