Exploring the Binding of GABA to the Insect RDL Receptor with Metadynamics

Abstract

The binding and unbinding of neurotransmitters to pentameric ligand-gated ion channels (pLGICs) occur on time scales much longer that those typically accessible to standard simulation protocols, such as molecular dynamics. In recent years, a number of enhanced sampling techniques have been developed to overcome this obstacle, allowing to simulate rare events with affordable computational resources. In this work, we applied the enhanced sampling method metadynamics to investigate the binding mechanism of GABA to the RDL receptor, a prototypical insect pLGIC involved in insecticide resistance. We built homology models mimicking the extracellular domains of the RDL receptor and simulated GABA binding and unbinding events. We calculated the binding free energy surfaces in the wild-type RDL receptor and in two mutant forms, where the key polar residues for ligand binding, Arg111 and Glu204, were mutated to the neutral Ala. The results are in agreement with mutagenesis electrophysiology experiments, where these mutants were measured as non-functional. Our simulations allowed us to characterize GABA interaction network and the protein movements which occurred in the binding process: in particular we investigated the dynamical behaviour of loop C, a flexible structure that plays an important role when the the neurotransmitter approaches or leaves the binding pocket.