Bayesian network models identify cooperative GPCR:G protein interactions that contribute to G protein coupling.

Cooperative interactions in protein-protein interfaces demonstrate the interdependency or the linked network-like behavior and their effect on the coupling of proteins. Cooperative interactions also could cause ripple or allosteric effects at a distance in protein-protein interfaces. Although they are critically important in protein-protein interfaces, it is challenging to determine which amino acid pair interactions are cooperative. In this work, we have used Bayesian network modeling, an interpretable machine learning method, combined with molecular dynamics trajectories to identify the residue pairs that show high cooperativity and their allosteric effect in the interface of G protein-coupled receptor (GPCR) complexes with Gα subunits. Our results reveal six GPCR:Gα contacts that are common to the different Gα subtypes and show strong cooperativity in the formation of interface. Both the C terminus helix5 and the core of the G protein are codependent entities and play an important role in GPCR coupling. We show that a promiscuous GPCR coupling to different Gα subtypes, makes all the GPCR:Gα contacts that are specific to each Gα subtype (Gαs, Gαi, and Gαq). This work underscores the potential of data-driven Bayesian network modeling in elucidating the intricate dependencies and selectivity determinants in GPCR:G protein complexes, offering valuable insights into the dynamic nature of these essential cellular signaling components.

Stabilization of interdomain interactions in G protein α subunits as a determinant of Gαi subtype signaling specificity.

Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector that is poorly activated by Gαi2. In a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 with the corresponding D in Gαi1, largely rescues PRG activation and interactions with other potential Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gαi2 than Gαi1. Mutation of A230 to D in Gαi2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gαi1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by G protein-coupled receptors.

A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

The plasma membrane is enriched for receptors and signaling proteins that are accessible from the extracellular space for pharmacological intervention. Here we conducted a series of CRISPR screens using human cell surface proteome and integrin family libraries in multiple cancer models. Our results identified ITGAV (integrin αV) and its heterodimer partner ITGB5 (integrin ß5) as the essential integrin α/ß pair for cancer cell expansion. High-density CRISPR gene tiling further pinpointed the integral pocket within the ß-propeller domain of ITGAV for integrin αVß5 dimerization. Combined with in silico compound docking, we developed a CRISPR-Tiling-Instructed Computer-Aided (CRISPR-TICA) pipeline for drug discovery and identified Cpd_AV2 as a lead inhibitor targeting the ß-propeller central pocket of ITGAV. Cpd_AV2 treatment led to rapid uncoupling of integrin αVß5 and cellular apoptosis, providing a unique class of therapeutic action that eliminates the integrin signaling via heterodimer dissociation. We also foresee the CRISPR-TICA approach to be an accessible method for future drug discovery studies.

Therapeutic targeting Tudor domains in leukemia via CRISPR-Scan Assisted Drug Discovery.

Epigenetic dysregulation has been reported in multiple cancers including leukemias. Nonetheless, the roles of the epigenetic reader Tudor domains in leukemia progression and therapy remain unexplored. Here, we conducted a Tudor domain-focused CRISPR screen and identified SGF29, a component of SAGA/ATAC acetyltransferase complexes, as a crucial factor for H3K9 acetylation, ribosomal gene expression, and leukemogenesis. To facilitate drug development, we integrated the CRISPR tiling scan with compound docking and molecular dynamics simulation, presenting a generally applicable strategy called CRISPR-Scan Assisted Drug Discovery (CRISPR-SADD). Using this approach, we identified a lead inhibitor that selectively targets SGF29's Tudor domain and demonstrates efficacy against leukemia. Furthermore, we propose that the structural genetics approach used in our study can be widely applied to diverse fields for de novo drug discovery.

Oligo-PROTAC strategy for cell-selective and targeted degradation of activated STAT3.

Decoy oligodeoxynucleotides (ODNs) allow targeting undruggable transcription factors, such as STAT3, but their limited potency and lack of delivery methods hampered translation. To overcome these challenges, we conjugated a STAT3-specific decoy to thalidomide, a ligand to cereblon in E3 ubiquitin ligase complex, to generate a proteolysis-targeting chimera (STAT3DPROTAC). STAT3DPROTAC downregulated STAT3 in target cells, but not STAT1 or STAT5. Computational modeling of the STAT3DPROTAC ternary complex predicted two surface lysines, K601 and K626, in STAT3 as potential ubiquitination sites. Accordingly, K601/K626 point mutations in STAT3, as well as proteasome inhibition or cereblon deletion, alleviated STAT3DPROTAC effect. Next, we conjugated STAT3DPROTAC to a CpG oligonucleotide targeting Toll-like receptor 9 (TLR9) to generate myeloid/B cell-selective C-STAT3DPROTAC. Naked C-STAT3DPROTAC was spontaneously internalized by TLR9+ myeloid cells, B cells, and human and mouse lymphoma cells but not by T cells. C-STAT3DPROTAC effectively decreased STAT3 protein levels and also STAT3-regulated target genes critical for lymphoma cell proliferation and/or survival (BCL2L1, CCND2, and MYC). Finally, local C-STAT3DPROTAC administration to human Ly3 lymphoma-bearing mice triggered tumor regression, while control C-STAT3D and C-SCR treatments had limited effects. Our results underscore the feasibility of using a PROTAC strategy for cell-selective, decoy oligonucleotide-based STAT3 targeting of and potentially other tumorigenic transcription factors for cancer therapy.

Cellular Lipids Regulate the Conformational Ensembles of the Disordered Intracellular Loop 3 in ß2 Adrenergic Receptor.

The structurally disordered intracellular loops (ICLs) of G protein-coupled receptors (GPCRs) play a critical role in G protein coupling. In our previous work, we used a combination of FRET-based and computational methodologies to show that the third intracellular loop (ICL3) modulates the activity and G protein coupling selectivity in GPCRs. In the current study, we have uncovered the role of several lipid components in modulating the conformational ensemble of ICL3 of the ß2-adrenergic receptor (ß2AR). Our findings indicate that phosphatidylinositol 4,5-bisphosphate (PIP2) in the inner leaflet of the membrane bilayer acts as a stabilizing anchor for ICL3, opening the intracellular cavity to facilitate G protein coupling. This interaction between PIP2 and ICL3 causes tilting of ß2AR within the cellular membrane. Notably, this tilting of the receptor is supported by ganglioside GM3 stabilizing the extracellular loops on the outer leaflet of the bilayer, thereby exerting an allosteric effect on the orthosteric ligand binding pocket. Our results underscore the significance of lipids in modulating GPCR activity, proposing an allosteric mechanism that occurs through the receptor's orientation within the membrane.

Molecular annotation of G protein variants in a neurological disorder.

Heterotrimeric G proteins transduce extracellular chemical messages to generate appropriate intracellular responses. Point mutations in GNAO1, encoding the G protein αo subunit, have been implicated in a pathogenic condition characterized by seizures, movement disorders, intellectual disability, and developmental delay (GNAO1 disorder). However, the effects of these mutations on G protein structure and function are unclear. Here, we report the effects of 55 mutations on Gαo conformation, thermostability, nucleotide binding, and hydrolysis, as well as interaction with Gßγ subunits, receptors, and effectors. Our effort reveals four functionally distinct groups of mutants, including one group that sequesters receptors and another that sequesters Gßγ, both acting in a genetically dominant manner. These findings provide a more comprehensive understanding of disease-relevant mutations and reveal that GNAO1 disorder is likely composed of multiple mechanistically distinct disorders that will likely require multiple therapeutic strategies.

Engineered odorant receptors illuminate structural principles of odor discrimination.

A central challenge in olfaction is understanding how the olfactory system detects and distinguishes odorants with diverse physicochemical properties and molecular configurations. Vertebrate animals perceive odors via G protein-coupled odorant receptors (ORs). In humans, ~400 ORs enable the sense of smell. The OR family is composed of two major classes: Class I ORs are tuned to carboxylic acids while Class II ORs, representing the vast majority of the human repertoire, respond to a wide variety of odorants. How ORs recognize chemically diverse odorants remains poorly understood. A fundamental bottleneck is the inability to visualize odorant binding to ORs. Here, we uncover fundamental molecular properties of odorant-OR interactions by employing engineered ORs crafted using a consensus protein design strategy. Because such consensus ORs (consORs) are derived from the 17 major subfamilies of human ORs, they provide a template for modeling individual native ORs with high sequence and structural homology. The biochemical tractability of consORs enabled four cryoEM structures of distinct consORs with unique ligand recognition properties. The structure of a Class I consOR, consOR51, showed high structural similarity to the native human receptor OR51E2 and yielded a homology model of a related member of the human OR51 family with high predictive power. Structures of three Class II consORs revealed distinct modes of odorant-binding and activation mechanisms between Class I and Class II ORs. Thus, the structures of consORs lay the groundwork for understanding molecular recognition of odorants by the OR superfamily.

Bayesian network models identify co-operative GPCR:G protein interactions that contribute to G protein coupling.

Cooperative interactions in protein-protein interfaces demonstrate the interdependency or the linked network-like behavior of interface interactions and their effect on the coupling of proteins. Cooperative interactions also could cause ripple or allosteric effects at a distance in protein-protein interfaces. Although they are critically important in protein-protein interfaces it is challenging to determine which amino acid pair interactions are cooperative. In this work we have used Bayesian network modeling, an interpretable machine learning method, combined with molecular dynamics trajectories to identify the residue pairs that show high cooperativity and their allosteric effect in the interface of G protein-coupled receptor (GPCR) complexes with G proteins. Our results reveal a strong co-dependency in the formation of interface GPCR:G protein contacts. This observation indicates that cooperativity of GPCR:G protein interactions is necessary for the coupling and selectivity of G proteins and is thus critical for receptor function. We have identified subnetworks containing polar and hydrophobic interactions that are common among multiple GPCRs coupling to different G protein subtypes (Gs, Gi and Gq). These common subnetworks along with G protein-specific subnetworks together confer selectivity to the G protein coupling. This work underscores the potential of data-driven Bayesian network modeling in elucidating the intricate dependencies and selectivity determinants in GPCR:G protein complexes, offering valuable insights into the dynamic nature of these essential cellular signaling components.

Unraveling allostery within the angiotensin II type 1 receptor for Gαq and ß-arrestin coupling.

G protein-coupled receptors engage both G proteins and ß-arrestins, and their coupling can be biased by ligands and mutations. Here, to resolve structural elements and mechanisms underlying effector coupling to the angiotensin II (AngII) type 1 receptor (AT1R), we combined alanine scanning mutagenesis of the entire sequence of the receptor with pharmacological profiling of Gαq and ß-arrestin engagement to mutant receptors and molecular dynamics simulations. We showed that Gαq coupling to AT1R involved a large number of residues spread across the receptor, whereas fewer structural regions of the receptor contributed to ß-arrestin coupling regulation. Residue stretches in transmembrane domain 4 conferred ß-arrestin bias and represented an important structural element in AT1R for functional selectivity. Furthermore, we identified allosteric small-molecule binding sites that were enclosed by communities of residues that produced biased signaling when mutated. Last, we showed that allosteric communication within AT1R emanating from the Gαq coupling site spread beyond the orthosteric AngII-binding site and across different regions of the receptor, including currently unresolved structural regions. Our findings reveal structural elements and mechanisms within AT1R that bias Gαq and ß-arrestin coupling and that could be harnessed to design biased receptors for research purposes and to develop allosteric modulators.

Oligo-PROTAC strategy for cell-selective and targeted degradation of activated STAT3.

Decoy-oligodeoxynucleotides (D-ODNs) can target undruggable transcription factors, such as STAT3. However, challenges in D-ODN delivery and potency hampered their translation. To overcome these limitations, we conjugated STAT3-specific D-ODN to thalidomide (Tha), a known ligand to cereblon (CRBN, a component of E3 ubiquitin ligase) to generate a proteolysis-targeting chimera (STAT3D PROTAC ). STAT3D PROTAC downregulated STAT3, but not STAT1 or STAT5, in target cells. Computational modeling of the STAT3D PROTAC ternary complex predicted two surface lysines on STAT3, K601 and K626 as potential ubiquitination sites for the PROTAC bound E3 ligase. Accordingly, K601/K626 point mutations in STAT3, as well as proteasome inhibitors, and CRBN deletion alleviated STAT3D PROTAC effect. Next, we conjugated STAT3D PROTAC to a CpG ligand targeting Toll-like receptor 9 (TLR9) to generate myeloid/B-cell-selective C-STAT3D PROTAC conjugate. Naked C-STAT3D PROTAC was spontaneously internalized by TLR9 + myeloid cells, B cells as well as human Ly18 and mouse A20 lymphoma cells, but not by T cells. C-STAT3D PROTAC decreased STAT3 levels to 50% at 250 nM and over 85% at 2 µM dosing in myeloid cells. We also observed significantly improved downregulation of STAT3 target genes involved in lymphoma cell proliferation and/or survival ( BCL2L1, CCND2, MYC ). Finally, we assessed the antitumor efficacy of C-STAT3D PROTAC compared to C-STAT3D or scrambled control (C-SCR) against human lymphoma xenotransplants. Local C-STAT3D PROTAC administration triggered lymphoma regression while control treatments had limited effects. Our results underscore feasibility of using PROTAC strategy for cell-selective, decoy oligonucleotide-based targeting of STAT3 and potentially other tumorigenic transcription factors for cancer therapy.

Molecular recognition of an aversive odorant by the murine trace amine-associated receptor TAAR7f.

There are two main families of G protein-coupled receptors that detect odours in humans, the odorant receptors (ORs) and the trace amine-associated receptors (TAARs). Their amino acid sequences are distinct, with the TAARs being most similar to the aminergic receptors such as those activated by adrenaline, serotonin and histamine. To elucidate the structural determinants of ligand recognition by TAARs, we have determined the cryo-EM structure of a murine receptor, mTAAR7f, coupled to the heterotrimeric G protein Gs and bound to the odorant N,N-dimethylcyclohexylamine (DMCH) to an overall resolution of 2.9 Å. DMCH is bound in a hydrophobic orthosteric binding site primarily through van der Waals interactions and a strong charge-charge interaction between the tertiary amine of the ligand and an aspartic acid residue. This site is distinct and non-overlapping with the binding site for the odorant propionate in the odorant receptor OR51E2. The structure, in combination with mutagenesis data and molecular dynamics simulations suggests that the activation of the receptor follows a similar pathway to that of the ß-adrenoceptors, with the significant difference that DMCH interacts directly with one of the main activation microswitch residues.

Stabilization of Interdomain Interactions in G protein αi Subunits Determines Gαi Subtype Signaling Specificity.

Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet the functional properties of these proteins with respect to GDP/GTP binding and regulation of adenylate cyclase are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector, however, it is poorly activated by Gαi2. Here, in a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 to the corresponding D in Gαi1, largely rescues PRG activation and interactions with other Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, dynamic separation at the HD-Ras-like domain (RLD) interface is prevalent in Gαi2 relative to Gαi1 and that mutation of A230s4h3.3 to D in Gαi2 stabilizes HD-RLD interactions through formation of an ionic interaction with R145HD.11 in the HD. These interactions in turn modify the conformation of Switch III. These data support a model where D229s4h3.3 in Gαi1 interacts with R144HD.11 stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to form the "ionic lock" to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by GPCRs.

Dynamic phosphatase-recruitment controls B-cell selection and oncogenic signaling.

Initiation of B-cell receptor (BCR) 1 signaling, and subsequent antigen-encounter in germinal centers 2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) 4 and lymphoma 5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells 6-9 , the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ 10 - 12 , SHIP1 13,14 and SHP1 14, 15,16 , conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268 ). Genetic rescue experiments identified CD25-S 268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25 S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca 2+ -oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.

Structural basis of odorant recognition by a human odorant receptor.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome1-3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.

Autoregulation of GPCR signalling through the third intracellular loop.

The third intracellular loop (ICL3) of the G protein-coupled receptor (GPCR) fold is important for the signal transduction process downstream of receptor activation1-3. Despite this, the lack of a defined structure of ICL3, combined with its high sequence divergence among GPCRs, complicates characterization of its involvement in receptor signalling4. Previous studies focusing on the ß2 adrenergic receptor (ß2AR) suggest that ICL3 is involved in the structural process of receptor activation and signalling5-7. Here we derive mechanistic insights into the role of ICL3 in ß2AR signalling, observing that ICL3 autoregulates receptor activity through a dynamic conformational equilibrium between states that block or expose the receptor's G protein-binding site. We demonstrate the importance of this equilibrium for receptor pharmacology, showing that G protein-mimetic effectors bias the exposed states of ICL3 to allosterically activate the receptor. Our findings additionally reveal that ICL3 tunes signalling specificity by inhibiting receptor coupling to G protein subtypes that weakly couple to the receptor. Despite the sequence diversity of ICL3, we demonstrate that this negative G protein-selection mechanism through ICL3 extends to GPCRs across the superfamily, expanding the range of known mechanisms by which receptors mediate G protein subtype selective signalling. Furthermore, our collective findings suggest ICL3 as an allosteric site for receptor- and signalling pathway-specific ligands.

A Closer Look at EGFR Inhibitor Resistance in Non-Small Cell Lung Cancer through the Lens of Precision Medicine.

The development of EGFR small-molecule inhibitors has provided significant benefit for the affected patient population. Unfortunately, current inhibitors are no curative therapy, and their development has been driven by on-target mutations that interfere with binding and thus inhibitory activity. Genomic studies have revealed that, in addition to these on-target mutations, there are also multiple off-target mechanisms of EGFR inhibitor resistance and novel therapeutics that can overcome these challenges are sought. Resistance to competitive 1st-generation and covalent 2nd- and 3rd-generation EGFR inhibitors is overall more complex than initially thought, and novel 4th-generation allosteric inhibitors are expected to suffer from a similar fate. Additional nongenetic mechanisms of resistance are significant and can include up to 50% of the escape pathways. These potential targets have gained recent interest and are usually not part of cancer panels that look for alterations in resistant patient specimen. We discuss the duality between genetic and nongenetic EGFR inhibitor drug resistance and summarize current team medicine approaches, wherein clinical developments, hand in hand with drug development research, drive potential opportunities for combination therapy.

Catalytic site mutations confer multiple states of G protein activation.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) that function as molecular switches for cellular growth and metabolism are activated by GTP and inactivated by GTP hydrolysis. In uveal melanoma, a conserved glutamine residue critical for GTP hydrolysis in the G protein α subunit is often mutated in Gαq or Gα11 to either leucine or proline. In contrast, other glutamine mutations or mutations in other Gα subtypes are rare. To uncover the mechanism of the genetic selection and the functional role of this glutamine residue, we analyzed all possible substitutions of this residue in multiple Gα isoforms. Through cell-based measurements of activity, we showed that some mutants were further activated and inactivated by G protein-coupled receptors. Through biochemical, molecular dynamics, and nuclear magnetic resonance-based structural studies, we showed that the Gα mutants were functionally distinct and conformationally diverse, despite their shared inability to hydrolyze GTP. Thus, the catalytic glutamine residue contributes to functions beyond GTP hydrolysis, and these functions include subtype-specific, allosteric modulation of receptor-mediated subunit dissociation. We conclude that G proteins do not function as simple on-off switches. Rather, signaling emerges from an ensemble of active states, a subset of which are favored in disease and may be uniquely responsive to receptor-directed ligands.

Biased agonists differentially modulate the receptor conformation ensembles in Angiotensin II type 1 receptor.

The structural features that contribute to the efficacy of biased agonists targeting G protein-coupled receptors (GPCRs) towards G proteins or ß-arrestin (ß-arr) signaling pathways is nebulous, although such knowledge is critical in designing biased ligands. The dynamics of the agonist-GPCR complex is one of the critical factors in determining agonist bias. Angiotensin II type I receptor (AT1R) is an ideal model system to study the molecular basis of bias since it has multiple ß-arr2 and Gq protein biased agonists as well as experimentally solved three dimensional structures. Using Molecular Dynamics (MD) simulations for the Angiotensin II type I receptor (AT1R) bound to ten different agonists, we infer that the agonist bound receptor samples conformations with different relative weights, from both the inactive and active state ensembles of the receptor. This concept is perhaps extensible to other class A GPCRs. Such a weighted mixed ensemble recapitulates the inter-residue distance distributions measured for different agonists bound AT1R using DEER experiments. The ratio of the calculated relative strength of the allosteric communication to ß-arr2 vs Gq coupling sites scale similarly to the experimentally measured bias factors. Analysis of the inter-residue distance distributions of the activation microswitches involved in class A GPCR activation suggests that ß-arr2 biased agonists turn on different combination of microswitches with different relative strengths of activation. We put forth a model that activation microswitches behave like rheostats that tune the relative efficacy of the biased agonists toward the two signaling pathways. Finally, based on our data we propose that the agonist specific residue contacts in the binding site elicit a combinatorial response in the microswitches that in turn differentially modulate the receptor conformation ensembles resulting in differences in coupling to Gq and ß-arrestin.