Exploring molecular mechanisms of ligand recognition by opioid receptors with metadynamics - PubMed (original) (raw)

Exploring molecular mechanisms of ligand recognition by opioid receptors with metadynamics

Davide Provasi et al. Biochemistry. 2009.

Abstract

Opioid receptors are G protein-coupled receptors (GPCRs) of utmost significance in the development of potent analgesic drugs for the treatment of severe pain. An accurate evaluation at the molecular level of the ligand binding pathways into these receptors may play a key role in the design of new molecules with more desirable properties and reduced side effects. The recent characterization of high-resolution X-ray crystal structures of non-rhodopsin GPCRs for diffusible hormones and neurotransmitters presents an unprecedented opportunity to build improved homology models of opioid receptors, and to study in more detail their molecular mechanisms of ligand recognition. In this study, possible pathways for entry of the nonselective antagonist naloxone (NLX) from the water environment into the well-accepted alkaloid binding pocket of a delta opioid receptor (DOR) molecular model based on the beta2-adrenergic receptor crystal structure are explored using microsecond-scale well-tempered metadynamics simulations. Using as collective variables distances that account for the position of NLX and of the receptor extracellular loop 2 in relation to the DOR binding pocket, we were able to distinguish between the different states visited by the ligand (i.e., docked, undocked, and metastable bound intermediates) and to predict a free energy of binding close to experimental values after correcting for possible drawbacks of the sampling approach. The strategy employed herein holds promise for its application to the docking of diverse ligands to the opioid receptors as well as to other GPCRs.

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Figures

Figure 1

Figure 1

Side view of the initial 3D model of human DOR built according to the procedure described in Methods. TM1, TM2, TM3, TM4, TM5, TM6, and TM7 are colored in purple, blue, light blue, light green, green, yellow, orange and red, respectively. The 5-degree conical restraint that was applied to circumscribe NLX sampling is indicated by the black lines defining the angle between the COM of the binding pocket, the COM of the ligand, and the COM of residue L3007.35 (black dot in the figure).

Figure 2

Figure 2

Free-energy surface of the NLX-DOR system reconstructed by well-tempered metadynamics as a function of the distance of the NLX COM (CV1) and of the distance of EL2 COM (CV2) from the binding pocket COM. Relevant states are labeled A (NLX bound into the well-accepted OR alkaloid binding pocket), B (NLX bound at the EL2/EL3 recognition cleft), and C (NLX at a metastable state in the helix bundle). Each contour represents a free-energy difference of 2 kcal/mol. The red solid line refers to the entry path obtained by NEB that was used to generate the entry path collective variables. Also represented are images of A, B1, B2, and C metastable states of DOR cut along their TM4 face and the position of NLX (black spheres) in the corresponding states.

Figure 3

Figure 3

Representative conformation extracted from the basin A of the free-energy surface showing NLX bound to the DOR well-accepted alkaloid binding pocket. Parts of TM2 and TM4 have been removed for clarity.

Figure 4

Figure 4

Representative conformations extracted from basins B1, B2 and C of the free-energy surface. Top (as seen from the extracellular side) and side views of NLX bound (A, B) to the EL2/EL3 cleft on the DOR surface in the conformation extracted from the B1 basin, (C, D) to the EL2/EL3 cleft on the DOR surface in the conformation extracted from the B2 basin, and (E, F) within the helix bundle, in the conformation extracted from the C basin.

Figure 5

Figure 5

The free-energy surface reconstructed using well-tempered metadynamics as a function of the position along (CV3) and the distance from (CV4) the suggested NLX entry path. Each contour represents a free-energy difference of 2 kcal/mol. Relevant states are labeled according to Figure 2, and are: A (NLX bound to the DOR well-accepted alkaloid binding pocket, see Figure 3), B1 (NLX bound to the most external location on the EL2/EL3 cleft, see Figures 4A,B), B2 (NLX bound to a more stable position on the EL2/EL3 cleft, see Figures 4C,D) and C (NLX at a metastable state within the helix bundle).

Figure 6

Figure 6

Integration of the free-energy profile of Figure 2 over the CV2 variable (distance of the EL2 C198-W209 region from the receptor alkaloid binding pocket) reported as a function of the CV1 variable (distance of NLX from the receptor alkaloid binding pocket) in the 0<CV1<20 region. The free-energy profile was shifted so that the reference state corresponds to the most stable one.

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