Structural and energetic effects of A2A adenosine receptor mutations on agonist and antagonist binding

Abstract

To predict structural and energetic effects of point mutations on ligand binding is of considerable interest in biochemistry and pharmacology. This is not only useful in connection with site-directed mutagenesis experiments, but could also allow interpretation and prediction of individual responses to drug treatment. For G-protein coupled receptors systematic mutagenesis has provided the major part of functional data as structural information until recently has been very limited. For the pharmacologically important A(2A) adenosine receptor, extensive site-directed mutagenesis data on agonist and antagonist binding is available and crystal structures of both types of complexes have been determined. Here, we employ a computational strategy, based on molecular dynamics free energy simulations, to rationalize and interpret available alanine-scanning experiments for both agonist and antagonist binding to this receptor. These computer simulations show excellent agreement with the experimental data and, most importantly, reveal the molecular details behind the observed effects which are often not immediately evident from the crystal structures. The work further provides a distinct validation of the computational strategy used to assess effects of point-mutations on ligand binding. It also highlights the importance of considering not only protein-ligand interactions but also those mediated by solvent water molecules, in ligand design projects.

Figure 1. Chemical structures of ligands and overview of the A_2AAR orthosteric binding site.

Structures of the antagonist ZM241385 (A) and the agonist NECA (B) are depicted together with compounds used as radioligands in the experimental site-directed mutagenesis considered in this work (see Tables 1, 2). (C) ZM241385 (green sticks) and NECA (magenta sticks) overlaid in the orthosteric binding site , . Receptor residues examined by alanine scanning are shown as spheres for antagonist binding (green), agonist binding (magenta) or both agonist and antagonist binding (yellow).

Figure 2. Structure of the A_2AAR−ZM241385 complex and relative ligand binding free energies for mutants.

(A) Starting structure used for the FEP simulations with TM helices shown and colored according to a rainbow representation in anti-clockwise order (TM1 = blue → TM7 = red). Residues subjected alanine mutation are depicted in sticks, together with crystal water molecules mediating receptor-ligand interactions, and dashed lines indicate hydrogen bonds. (B) Calculated (gray bars) and experimental (black bars) relative binding free energies (kcal/mol) for ZM241385 to the fourteen A_2AAR alanine mutants compared to the wt receptor. The star symbol denotes that an experimental value could not be determined and approximates the detection threshold of the experiment.

Figure 3. Correlation of ligand interaction energies with alanine mutations for the A_2AAR−ZM241385 complex.

(A) Correlation diagram showing the change in non-bonded interaction energies (electrostatic – top, van der Waals – bottom) for relevant binding site residues (y-axis) upon a given alanine mutation (x-axis). Only residues with any absolute interaction energy change above 1 kcal/mol are shown, where the water molecules are present throughout the MD simulations and also observed in the crystal structure. (B) The corresponding affected residues and water molecules are shown in sticks for the initial 3D structure of the complex.

Figure 4. Structure of the A_2AAR*−NECA complex and relative binding free energies for mutants.

(A) Starting structure used for the FEP simulations, with explicit representation of residues subjected to alanine mutation and crystal water molecules as in Figure 2. (B) Calculated (light gray bars) and experimental (black bars) relative binding free energies (kcal/mol) for the seventeen A_2AAR^* alanine mutants compared to the wt receptor. The star symbol denotes that an experimental value could not be determined and approximates the detection threshold of the experiment.

Figure 5. Effect of mutating a residue in the residue-ligand interaction energies for the A_2AAR−NECA complex.

(A) Correlation diagram showing the change in non-bonded interaction energies (electrostatic – top, van der Waals – bottom) for relevant binding site residues (y-axis) upon a given alanine mutation (x-axis). Only residues with any absolute interaction energy change above 1 kcal/mol are shown, where the water molecules are present throughout the MD simulations and also observed in the crystal structure. (B) The corresponding affected residues and water molecules are shown in sticks for the initial 3D structure of the complex.

Figure 6. Structural effects of the T88^3.36A mutation on agonist binding.

The average MD conformation of the T88^3.36A mutant NECA complex with interacting sidechains and water molecules shown (solid sticks) overlaid on the initial wt conformation (transparent sticks).