Prediction of allosteric sites on protein surfaces with an elastic-network-model-based thermodynamic method

Allostery is a rapid and efficient way in many biological processes to regulate protein functions, where binding of an effector at the allosteric site alters the activity and function at a distant active site. Allosteric regulation of protein biological functions provides a promising strategy for novel drug design. However, how to effectively identify the allosteric sites remains one of the major challenges for allosteric drug design. In the present work, a thermodynamic method based on the elastic network model was proposed to predict the allosteric sites on the protein surface. In our method, the thermodynamic coupling between the allosteric and active sites was considered, and then the allosteric sites were identified as those where the binding of an effector molecule induces a large change in the binding free energy of the protein with its ligand. Using the proposed method, two proteins, i.e., the 70 kD heat shock protein (Hsp70) and GluA2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, were studied and the allosteric sites on the protein surface were successfully identified. The predicted results are consistent with the available experimental data, which indicates that our method is a simple yet effective approach for the identification of allosteric sites on proteins.

Figure 1

(a) The crystal structure of Hsp70 from Escherichia coli (PDB code: 4JNE), which is composed of two domains, i.e., NBD and SBD. The NBD consists of four subdomains: IA displayed as red (dark gray) solid ribbon, IB shown as yellow (light gray) solid ribbon, IIA displayed as yellow (light gray) flat ribbon, and IIB shown as red (dark gray) flat ribbon, where IA and IB form lobe I, and IIA and IIB form lobe II. The SBD contains two subdomains: SBDα displayed as purple (light gray) line ribbon and SBDβ shown as red (dark gray) tube. (b) The generated 195 surface points by using the msms program, which are shown as black balls in the figure. The ATP molecular binds to the nucleotide-binding site in NBD, which is shown in the black stick model.

Figure 2

$\Delta \Delta G$ values for all the perturbed surface points. The points with the absolute value of $\Delta \Delta G$ larger than 0.002 were identified as the allosteric sites. The dashed line in the figure corresponds to the absolute value of $\Delta \Delta G$ to be 0.002. In this figure, the unit of $\Delta \Delta G$ is $\frac{3}{2}{k}_{B}T$.

Figure 3

The locations of the allosteric sites identified by our method. The predicted allosteric sites are grouped into five groups, which are marked by black ellipses and denoted by the numbers 1–5 in the figure.

Figure 4

(a) The x-ray crystal structure of the full-length rat GluA2 AMPA receptor in the resting state (PDB code: 3KG2), which is composed of four subunits, i.e., subunits A shown as red (dark gray) solid ribbon, B shown as yellow (light gray) solid ribbon, C displayed as yellow (light gray) tube, and D displayed as red (dark gray) tube. These four subunits are assembled as a dimer of dimers. Each subunit consists of four domains, which are NTD, LBD, TMD, and CTD. CTD is not resolved in the crystal structure. (b) The constructed dimeric complex structure of AMPA receptor where the ligand glutamate binds to subunit A. The glutamate is shown as a stick in black. Based on the constructed structure of AMPA receptor, surface points were generated by using the msms program and a total of 500 surface points were randomly selected, which are shown as black balls in the figure.

Figure 5

$\Delta \Delta G$ values for all the 500 perturbed surface points. The surface points with the absolute value of $\Delta \Delta G$ larger than 0.003 were identified as the allosteric sites. The dashed line in the figure corresponds to the absolute value of $\Delta \Delta G$ to be 0.003. In this figure, the unit of $\Delta \Delta G$ is $\frac{3}{2}{k}_{B}T$.

Figure 6

The locations of the allosteric sites identified by our method. The region of the structure marked by the rectangle is enlarged on the right. The predicted allosteric sites are grouped into five regions, which are marked by black ellipses and denoted by the numbers 1–5 in the figure.