Insights from free-energy calculations: protein conformational equilibrium, driving forces, and ligand-binding modes

Abstract

Accurate free-energy calculations provide mechanistic insights into molecular recognition and conformational equilibrium. In this work, we performed free-energy calculations to study the thermodynamic properties of different states of molecular systems in their equilibrium basin, and obtained accurate absolute binding free-energy calculations for protein-ligand binding using a newly developed M2 algorithm. We used a range of Asp-Phe-Gly (DFG)-in/out p38α mitogen-activated protein kinase inhibitors as our test cases. We also focused on the flexible DFG motif, which is closely connected to kinase activation and inhibitor binding. Our calculations explain the coexistence of DFG-in and DFG-out states of the loop and reveal different components (e.g., configurational entropy and enthalpy) that stabilize the apo p38α conformations. To study novel ligand-binding modes and the key driving forces behind them, we computed the absolute binding free energies of 30 p38α inhibitors, including analogs with unavailable experimental structures. The calculations revealed multiple stable, complex conformations and changes in p38α and inhibitor conformations, as well as balance in several energetic terms and configurational entropy loss. The results provide relevant physics that can aid in designing inhibitors and understanding protein conformational equilibrium. Our approach is fast for use with proteins that contain flexible regions for structure-based drug design.

Figure 1

Overall view of the p38α structure. (a) Two inhibitors, SB218655 (red) and urea 16 (blue), bind to p38α. The flexible and rigid sets are shown in yellow and green, respectively. (b) The activation loop can adopt both DFG-in (red, PDB 1a9u) and DFG-out (blue, PDB 1w82) states for ligand binding.

Figure 2

Sampled conformations of free DFG-in and DFG-out p38α. (A1 and A2) Overview of DFG-in and DFG-out conformations, respectively. The crystal structure is in blue (see Fig. 1 for PDB codes), the sampled conformation with the lowest free energy is in red, and conformations with energies within 10RT above the global energy minimum are in pink. (B1) Phe-169 (bond form) is buried in the hydrophobic cluster in a DFG-in complex and the activation loop is exposed to the solvent (colored thin line). The red and light blue tubes represent DFG-in and DFG-out conformations, respectively. (B2) Side-chain arrangements in the DFG-out state. Key interactions between important atom pairs are shown by the pink dashed line. The light red and blue tubes represent DFG-in and DFG-out conformations, respectively.

Figure 3

Positions and motions of key residues in DFG-out p38α. (a) Orange spheres: nonpolar spine residues; yellow spheres: nonpolar clusters (Tyr-35 and Leu-171); blue spheres: Asp-168–Gly-170 H-bond pair. (b and c) Low-energy conformations within 10RT of global energy minimum from computed DFG-in (b) and DFG-out (c) free p38α. Note that no H-bonds formed in the DFG-in state shown in b, and dashed lines in c indicate the H-bond between Asp-168 and Gly-170. Tubes and bonds in purple represent structures taken from PBD 1a9u (b) and 1w82 (c).

Figure 4

Calculated versus experimental binding free energies (kcal/mol) for p38α inhibitors.

Figure 5

Complex conformations of SB203580 and p38α with both DFG-in and DFG-out conformations. (a) Sampled ligand-binding modes (colored bond), DFG-in (red), and DFG-out (blue) conformations, which illustrate space for interconverting the activation loop. (b) Low-energy conformations within 10RT of the global energy minimum from the calculations, showing the fluctuation of Phe-169 in the DFG-out state and the highly flexible methylsulfinylphenyl group. Sampled DFG-in and DFG-out conformations are red and blue, respectively, and structures from PDB IDs 1a9u and 3gcp are orange and black, respectively. (c) The sampled activation loop and Phe-169 in both DFG-in and DFG-out conformations are in cyan; crystal structures with PDB IDs, 3hv7, 1w82, 3gcp, and 1a9u are in purple, yellow, black, and orange, respectively. Note that the structure in complex with SB203580 from PDB 3gcp is the Cys-172 mutant (black arrow) and shows different conformation (black) from our samples and other experimental structures.

Figure 6

Binding modes of pyrazolourea 1 and its analogs. (a) Stable charge interactions among Glu-71 and Asp-168 (red) and the ligand (cyan). (b) Global energy minimum found in the complex. The template pyrazolourea 1 is shown as a colored bond. Gray, purple, yellow, orange, and green represent the pyrazolourea 1 analogs (a), (b), (c), (d), and (e), respectively. The charge interactions among Glu-71, Asp-168, and the inhibitors are represented as dashed lines. (c) Low-energy conformations within 10RT of the global energy minimum of bound inhibitors. Each analog shows flexibility in different function groups; however, changes in configurational entropy upon binding cannot be easily predicted from looking at the ligand-binding modes.