Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2.

Activation of G protein-coupled receptors (GPCRs) is an allosteric process. It involves conformational coupling between the orthosteric ligand binding site and the G protein binding site. Factors that bind at non-cognate ligand binding sites to alter the allosteric activation process are classified as allosteric modulators and represent a promising class of therapeutics with distinct modes of binding and action. For many receptors, how modulation of signaling is represented at the structural level is unclear. Here, we developed fluorescence resonance energy transfer (FRET) sensors to quantify receptor modulation at each of the three structural domains of metabotropic glutamate receptor 2 (mGluR2). We identified the conformational fingerprint for several allosteric modulators in live cells. This approach enabled us to derive a receptor-centric representation of allosteric modulation and to correlate structural modulation to the standard signaling modulation metrics. Single-molecule FRET analysis revealed that a NAM (egative allosteric modulator) increases the occupancy of one of the intermediate states while a positive allosteric modulator increases the occupancy of the active state. Moreover, we found that the effect of allosteric modulators on the receptor dynamics is complex and depend on the orthosteric ligand. Collectively, our findings provide a structural mechanism of allosteric modulation in mGluR2 and suggest possible strategies for design of future modulators.

Mechanism of sensitivity modulation in the calcium-sensing receptor via electrostatic tuning.

Transfer of information across membranes is fundamental to the function of all organisms and is primarily initiated by transmembrane receptors. For many receptors, how ligand sensitivity is fine-tuned and how disease associated mutations modulate receptor conformation to allosterically affect receptor sensitivity are unknown. Here we map the activation of the calcium-sensing receptor (CaSR) - a dimeric class C G protein-coupled receptor (GPCR) and responsible for maintaining extracellular calcium in vertebrates. We show that CaSR undergoes unique conformational rearrangements compared to other class C GPCRs owing to specific structural features. Moreover, by analyzing disease associated mutations, we uncover a large permissiveness in the architecture of the extracellular domain of CaSR, with dynamics- and not specific receptor topology- determining the effect of a mutation. We show a structural hub at the dimer interface allosterically controls CaSR activation via focused electrostatic repulsion. Changes in the surface charge distribution of this hub, which is highly variable between organisms, finely tune CaSR sensitivity. This is potentially a general tuning mechanism for other dimeric receptors.

An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein.

The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme docking site in the Rb C-terminal domain that is required for efficient PP1c activity toward Rb. The phosphatase docking site overlaps with the known docking site for cyclin-dependent kinase (Cdk), and PP1 competition with Cdk-cyclins for Rb binding is sufficient to retain Rb activity and block cell-cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking.