The Mechanism of Ligand-Induced Activation or Inhibition of m- and k-Opioid Receptors **

G-protein-coupled receptors (GPCRs) are important targets for treating severe diseases. However why certain molecules act as activators whereas others, with similar structures, block GPCR activation, is poorly understood since the same molecule can activate one receptor subtype while blocking another closely related receptor. To shed light on these central questions, we used all-atom, long-time-scale molecular dynamics simulations on the k-opioid and m-opioid receptors (kOR and mOR). We found that water molecules penetrating into the receptor interior mediate the activating versus blocking effects of a particular ligand–receptor interaction. Both the size and the flexibility of the bound ligand regulated water influx into the receptor. The solvent-accessible inner surface area was found to be a parameter that can help predict the function of the bound ligand. The known crystal structures of ligand-free and ligand-bound G-protein-coupled receptors (GPCRs), as well as their ternary complexes with G proteins, show a large number of structural details . Based on static crystal structures and molecular dynamics simulations, some common elements in the activation mechanism of GPCRs have been found, including side-chain microswitches, movement of transmembrane helices, and rearrangement of internal water molecules. Herein, we concentrate on the question of whether specific properties of GPCR ligands can be predicted from the structural features of the corresponding ligand–receptor complexes obtained from computer simulations. Levallorphan is a morphine-like drug that is widely used as an antidote and opioid modulator. It acts as an agonist for k-opioid receptors (kORs) but as an antagonist for m-opioid receptors (mORs). As a result, it blocks the effects of stronger agents with greater intrinsic activity, such as morphine or endogenous b-endorphin. To address the different effects of levallorphan on kORs and mORs, we first performed all-atom molecular dynamics (MD) simulations on the crystal structures of both the kOR (pdb: 4DJH) and mOR (pdb: 4DKL) (Table 1). Our previous work indicated that a Na ion from bulk water can diffuse into the allosteric site of mOR, bind to the highly conserved D114, and thereby influence receptor function. To determine whether this finding would pertain to kOR, we performed 0.5 ms MD simulations for apo-kOR Table 1: Ligand–receptor systems used for MD simulations. Receptor kOR kOR–jdt kOR–lev mOR–lev mOR–mor Ligand – JDTic levallorphan levallorphan morphine Function apo antagonist agonist antagonist agonist