Dataset Information

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics.

ABSTRACT: G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/G? subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.

SUBMITTER: Zhang P

PROVIDER: S-EPMC4505487 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics.

Zhang Ping P Leger Andrew J AJ Baleja James D JD Rana Rajashree R Corlin Tiffany T Nguyen Nga N Koukos Georgios G Bohm Andrew A Covic Lidija L Kuliopulos Athan A

The Journal of biological chemistry 20150501 25

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, ...[more]

PMID: 25934391

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Project description:G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and ?-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the ?2-adrenergic receptor (?2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct ?2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for ?2AR in the presence of Nb80 compared to the affinity of isoprenaline for ?2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the ?2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

Project description:Metabotropic glutamate receptors (mGluR) are mainly expressed in the central nervous system (CNS) and contain eight receptor subtypes, named mGluR1 to mGluR8. The crystal structures of mGluR1 and mGluR5 that are bound with the negative allosteric modulator (NAM) were reported recently. These structures provide a basic model for all class C of G-protein coupled receptors (GPCRs) and may aid in the design of new allosteric modulators for the treatment of CNS disorders. However, these structures are only combined with NAMs in the previous reports. The conformations that are bound with positive allosteric modulator (PAM) or agonist of mGluR1/5 remain unknown. Moreover, the structural information of the other six mGluRs and the comparisons of the mGluRs family have not been explored in terms of their binding pockets, the binding modes of different compounds, and important binding residues. With these crystal structures as the starting point, we built 3D structural models for six mGluRs by using homology modeling and molecular dynamics (MD) simulations. We systematically compared their allosteric binding sites/pockets, the important residues, and the selective residues by using a series of comparable dockings with both the NAM and the PAM. Our results show that several residues played important roles for the receptors' selectivity. The observations of detailed interactions between compounds and their correspondent receptors are congruent with the specificity and potency of derivatives or compounds bioassayed in vitro. We then carried out 100 ns MD simulations of mGluR5 (residue 26-832, formed by Venus Flytrap domain, a so-called cysteine-rich domain, and 7 trans-membrane domains) bound with antagonist/NAM and with agonist/PAM. Our results show that both the NAM and the PAM seemed stable in class C GPCRs during the MD. However, the movements of "ionic lock," of trans-membrane domains, and of some activation-related residues in 7 trans-membrane domains of mGluR5 were congruent with the findings in class A GPCRs. Finally, we selected nine representative bound structures to perform 30 ns MD simulations for validating the stabilities of interactions, respectively. All these bound structures kept stable during the MD simulations, indicating that the binding poses in this present work are reasonable. We provided new insight into better understanding of the structural and functional roles of the mGluRs family and facilitated the future structure-based design of novel ligands of mGluRs family with therapeutic potential.

Dataset Information

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics.

Publications

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics.

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