Extended Data Fig. 5: Effects of interface mutations on clamshell closure.
a, smFRET distributions for LBD closure for two mutants demonstrate that R177A, but not D95A, leads to a reduction in glutamate affinity, but that in the presence of a high-affinity agonist, both mutants populate fully closed states (mGluR2-WT data seen in Fig. 1 shown on the left for comparison; data presented as mean ± s.e.m.; 4–6 movies per histogram). b, In a crystal structure (PDB ID: 4XAQ) of the mGluR2 LBD, R177 adopts two distinct orientations. We speculate that the nonequivalent effects of D95A and R177A on ligand affinity and intersubunit twisting are due, at least in part, to the ability of R177 to engage R243 on the opposite protomer via a π–π stack, one of the two R177 orientations observed in the crystal structure of agonist-bound mGluR2 LBD (PDB ID: 4XAQ). Classical molecular mechanics force fields do not explicitly represent π–π interactions, suggesting that our simulations may over-represent the orientation of R177 that engages D95. Additionally, introduction of R243A in single-molecule constructs substantially reduced expression, preventing smFRET investigation. c, Conservation of the interface network across the eight mGluR subtypes found in Rattus norvegicus.
