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

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.

Exploring the Activation Mechanism of the mGlu5 Transmembrane Domain

“…by "a" according to Ballesteros-Weinstein scheme (Ballesteros and Weinstein, 1995)] for class A GPCRs or followed by "c" according to Pin et al (2003) scheme for class C GPCRs). Despite previous studies have proposed functional similarities between class A GPCR micro-switches and certain non-homologous class C GPCR residues (Perez-Benito et al, 2017; Llinas Del Torrent et al, 2019) their respective conformational changes have been observed only in MD simulations of mGlu receptor homology models, but not in mGlu receptor crystal structures (Dore et al, 2014;Christopher et al, 2015;Koehl et al, 2019) or in MD simulations starting from those (Feng et al, 2015;Dalton et al, 2017;Perez-Benito et al, 2017;Llinas Del Torrent et al, 2019). Furthermore, while the outward movement of TM6 is a hallmark of class A GPCR activation, the extent of this movement is noticeably lower in mGlu5 even when enhanced sampling methods are employed (Cong et al, 2019).…”

“…In particular, a region was detected (blue circle, top of Figure 3) which includes a critical point that appears to identify a higher-energy "pseudo-stable" receptor state. It could be hypothesized that this region corresponds to an active-like receptor conformation on the basis that separation of intracellular loops is predicted to be a necessary feature of mGlu receptor activation (Dalton et al, 2017). Interestingly, the PMF whose collective variable involves the explicit breaking of the Lys665-Glu770 ionic lock did not show any clear minimum along its trajectory.…”

“…This is potentially in agreement with previous studies that showed that the breaking of the ionic lock is not a sufficient condition for GPCR activation (Dror et al, 2009;Lans et al, 2015b) because it is possible to find some inactive Class A GPCR structures with a broken ionic lock [see for instance A2AR PDB ID: 3EML (Jaakola et al, 2008) and β2AR PDB ID: 2RH1 (Cherezov et al, 2007)]. Furthermore, in MD simulations revealing ligandmediated mGlu4 conformational change, it was found that receptor "activation" resulted in transient disruption of the ionic lock but not a clear break (Dalton et al, 2017).…”

“…Significant efforts have been made to decipher the mGlu5 receptor structure-activity translation machinery (Kaae et al, 2012;Dalton et al, 2014Dalton et al, , 2017Gregory et al, 2014;Gomez-Santacana et al, 2017;Cong et al, 2019;Jojart et al, 2019;Llinas Del Torrent et al, 2019). In general, a delicate moleculargear receptor system whose mechanism of action is not yet well understood has become apparent.…”

“…To take a step forward in previous MD simulations of the mGlu5 receptor (Dalton et al, 2016(Dalton et al, , 2017 we provide herein potential of mean force (PMF) calculations (using umbrella sampling) linked to MD simulations of the TM domain of this receptor in its apo form. The present computational study, although conceived under a reductionist approach because it includes only one TM domain of a dimeric 3-domain receptor, has allowed the identification of some conformational features that can help to understand the intricacies of mGlu activation mechanism.…”