Analysis of positive and negative allosteric modulation in metabotropic glutamate receptors 4 and 5 with a dual ligand

James A R Dalton,Jean-Philippe Pin,Jesús Giraldo

doi:10.1038/s41598-017-05095-5

Abstract

As class C GPCRs and regulators of synaptic activity, human metabotropic glutamate receptors (mGluRs) 4 and 5 are prime targets for allosteric modulation, with mGlu5 inhibition or mGlu4 stimulation potentially treating conditions like chronic pain and Parkinson’s disease. As an allosteric modulator that can bind both receptors, 2-Methyl-6-(phenylethynyl)pyridine (MPEP) is able to negatively modulate mGlu5 or positively modulate mGlu4. At a structural level, how it elicits these responses and how mGluRs undergo activation is unclear. Here, we employ homology modelling and 30 µs of atomistic molecular dynamics (MD) simulations to probe allosteric conformational change in mGlu4 and mGlu5, with and without docked MPEP. Our results identify several structural differences between mGlu4 and mGlu5, as well as key differences responsible for MPEP-mediated positive and negative allosteric modulation, respectively. A novel mechanism of mGlu4 activation is revealed, which may apply to all mGluRs in general. This involves conformational changes in TM3, TM4 and TM5, separation of intracellular loop 2 (ICL2) from ICL1/ICL3, and destabilization of the ionic-lock. On the other hand, mGlu5 experiences little disturbance when MPEP binds, maintaining its inactive state with reduced conformational fluctuation. In addition, when MPEP is absent, a lipid molecule can enter the mGlu5 allosteric pocket.

Highlights

With eight subtypes, human metabotropic glutamate receptors are involved in the modulation of preand postsynaptic neuronal activity through the binding of glutamate, the major excitatory neurotransmitter in the CNS1, 2
We gain two benefits: first, we avoid the structure-activity noise that is present in ligand space associated with metabotropic glutamate receptors (mGluRs) and second, we focus on receptor recognition modes that may discriminate between positive allosteric modulator (PAM) and negative allosteric modulator (NAM) ligand functions
transmembrane helices (TMs) domain crystal structure were added as described in Methods, mutant residues reverted back to wt, and co-crystallized mavoglurant removed from the allosteric binding-site, generating an apo receptor state

Summary

Introduction

Human metabotropic glutamate receptors (mGluRs) are involved in the modulation of preand postsynaptic neuronal activity through the binding of glutamate, the major excitatory neurotransmitter in the CNS1, 2. These modelling studies, as well as mGlu[1] and mGlu[5] crystal structures, reveal little about the activation process of mGluRs or specific mechanisms behind positive allosteric modulation This is further complicated by mGluR activation occurring by two different means, either as homo-dimers in vivo[6], or single truncated TM domains in vitro[5]. Our current understanding of mGluR homo-dimer activation proceeds first by the binding of glutamate (or agonist) to extracellular domains causing an inter-domain scissoring movement, signal transmission through cysteine-rich domains, and sequential inter-subunit and intra-subunit conformational changes of TM domains inside the membrane[6, 34, 35] This process leads to receptor activation and G-protein binding, but at only one of the TM domains, not both[6]. A mechanism of mGlu[4] TM domain receptor activation is suggested, which is perhaps applicable to all mGluRs and is supported by pre-existing experimental data[4, 5, 32, 35, 58,59,60,61,62]

Methods

Results

Discussion

Conclusion