Differences between G‐Protein‐Stabilized Agonist–GPCR Complexes and their Nanobody‐Stabilized Equivalents

Abstract

Protein nanobodies have been used successfully as surrogates for unstable G-proteins in order to crystallizeGprotein-coupled receptors (GPCRs) in their active states.W e used molecular dynamics (MD) simulations,i ncluding metadynamics enhanced sampling, to investigate the similarities and differences between GPCR-agonist ternary complexes with the a-subunits of the appropriate G-proteins and those with the protein nanobodies (intracellular binding partners,IBPs) used for crystallization. In two of the three receptors considered, the agonist-binding mode differs significantly between the two alternative ternary complexes.T he ternary-complex model of GPCR activation entails enhancement of ligand binding by bound IBPs:O ur results showt hat IBP-specific changes can alter the agonist binding modes and thus also the criteria for designing GPCR agonists.

Mechanistic Insights into Specific G Protein Interactions with Adenosine Receptors Revealed by Accelerated Molecular Simulations

“…In contrast, residue Asp316 (Gh4s6.9) of the Gi protein formed polar interactions with residues Lys 6. 25 and Tyr 6.26 in the A1AR (Figure 5A).…”

“…17 Metadynamics simulations have been performed to investigate the dynamic effects of different GPCR ligands and intracellular binding partners 24 and examine differences of GPCRs coupled by the G protein versus its mimetic nanobody. 25 Nevertheless, these enhanced simulation methods require predefined collective variables and may apply constrains on the conformational space of the proteins. In this regard, a novel and robust Gaussian accelerated MD (GaMD) method has been developed to allow for unconstrained enhanced sampling and free energy calculations of large biomolecules [26][27][28] .…”

Mechanistic insights into dopaminergic and serotonergic neurotransmission – concerted interactions with helices 5 and 6 drive the functional outcome

“…The power of enhanced sampling techniques for capturing biologically relevant events has been shown in previous studies. [17][18][19] Here, we use this approach to construct the complete energetic binding landscape of dopamine and closely related signaling probes. The small size and low number of rotatable bonds of studied compounds allow for an exhaustive sampling of their binding.…”

Universal Activation Index for Class A GPCRs

“…We used these data to construct a local activity model for H4R, 7 but data on a single receptor alone is not sufficient for constructing a generally applicable model. We therefore used data from a further 10 published simulations, 5,6,8 for which binary active/inactive assignments are possible. We previously used these simulations to investigate the similarities and differences between GPCR ternary (agonist-GPCR-IBP) complexes with G-protein and β-arrestin IBPs.…”