The folded structure of a protein is understood to be an optimal energy state. However, previous studies have shown that certain amino acid residue positions that play a critical role in protein function are often in a suboptimal energy state or "frustrated". Here, we leverage over 1200 three-dimensional structures of G protein-coupled receptors (GPCRs) to demonstrate that residues at the interface between GPCR and its ligand or G protein contain a higher density of frustrated residues compared to other structural regions in the receptor. Likewise, the G alpha subunit of the trimeric G proteins shows multiple clusters of highly frustrated residues on its surface that overlap with their effector protein (Gb gamma, RGS, Adenylyl cyclase, Ric8) binding interfaces. Our study highlights the use of protein frustration as one of the multiple structural properties to identify protein-protein interfaces and for prospective prediction of potential ligand binding sites.

The folded structure of a protein is understood to be an optimal energy state. However, previous studies have shown that certain amino acid residue positions that play a critical role in protein function are often in a suboptimal energy state or "frustrated". Here, we leverage over 1200 three-dimensional structures of G protein-coupled receptors (GPCRs) to demonstrate that residues at the interface between GPCR and its ligand or G protein contain a higher density of frustrated residues compared to other structural regions in the receptor. Likewise, the Gα subunit of the trimeric G proteins shows multiple clusters of highly frustrated residues on its surface that overlap with their effector protein (Gbγ, RGS, Adenylyl cyclase, Ric8) binding interfaces. Our study highlights the use of protein frustration as one of the multiple structural properties to identify protein-protein interfaces and for prospective prediction of potential ligand binding sites.

Glucose-dependent insulinotropic peptide receptor (GIPR) stimulates insulin release and regulates metabolic homeostasis. GIPR function is shaped by spatiotemporal trafficking of this G protein-coupled receptor (GPCR). While GPCR endocytosis is traditionally associated with β-arrestin, GIPR internalization is only modestly dependent on this pathway. In this study, we demonstrate that GIPR engages a cytoskeletal motor, myosin VI to drive receptor endocytosis. GIPR engages the adaptor-motor complex through a PDZ-binding motif (PBM) at its C-ail. Interestingly, β-arrestin binding to phosphorylated residues upstream of the PBM enhance myosin VI recruitment and activation. GIPR internalization is dependent on both receptor phosphorylation and the PBM site to recruit β-arrestin and myosin VI, respectively. Cooperative engagement of β-arrestin and myosin VI results in desensitization of GIP-stimulated cAMP signaling while activating pERK1/2 from endosomal compartments. Blocking myosin VI activity enhances insulin release in pancreatic beta cells, demonstrating a novel role for this pathway in regulating the physiological effects of GIPR. Our findings highlight the direct convergence of two independent trafficking pathways at the level of the receptor C-tail, with implications for the nuanced regulation of individual GPCRs through the differential engagement of β-arrestin and myosin VI.

Despite extensive structural and functional studies, the molecular mechanisms governing G-protein coupled receptor-G (GPCR-G) protein coupling selectivity remain unresolved. Here, using an interpretable machine learning Bayesian Network model with Molecular Dynamics simulations and experiments, we reveal the influence of distant residue communities within the Gα protein core on coupling selectivity. We observed distinct cooperative hotspot residues across different Gα protein subtypes, including key regions such as the N-terminus, h4s6 loop, and H5 helix. These results demonstrate the intricate allosteric dependencies between the core and the H5 helix in stabilizing selective interactions. The functional significance of these cooperative regions is validated through subtype-swapping mutations. By introducing targeted Gαq-like mutations in the Gαs core, we successfully altered the receptor coupling profile to signal through Gαq. Our findings emphasize that cooperative interactions in the Gα core are not only crucial for selectivity but can also be leveraged to engineer Gα proteins with tailored coupling preferences.

GPCR therapeutics primarily target structured cavities formed by the seven transmembrane α-helices. However, amino acid sequence conservation in structured regions, especially among receptor subtypes, limits target selectivity. Here, we leverage the sequence divergence of the third intracellular loop (ICL3) of the receptor fold to derive a selective positive allosteric modulator for the β2 adrenergic receptor (β2AR). We repurpose the variable regions of a previously reported monoclonal antibody (Mab5) as single-chain (ScFv5) and single-domain (VhhL5) antibody fragments that enhance the maximal second messenger cyclic AMP signaling response downstream of the receptor. Despite ScFv5 binding a segment of ICL3 distinct to β2AR, it also non-selectively binds and modulates cAMP signaling downstream of β1AR and β3AR. We find that this lack of specificity stems from multiple redundant interactions that facilitate ScFv5-ICL3 binding. In contrast, VhhL5, derived from the Mab5 light chain, selectively modulates β2AR signaling over the β1/β3 subtypes. Mechanistically, VhhL5 enhances agonist-stimulated β2AR-G protein coupling through releasing autoinhibitory ICL3 conformational states. In parallel, VhhL5 decreases β2AR internalization promoting greater ligand-induced accumulation of cAMP. Our study demonstrates proof-of-concept for selective allosteric modulation of a GPCR by targeting a sequence divergent loop region within the receptor fold.

Titrating G protein-coupled receptor (GPCR) signaling to achieve desired physiological outcomes without overdose can be accomplished through partial agonism rather than drug dosage. In this study, we use FRET-based biosensors in combination with ordinary differential equation modelling to identify two key kinetic features that determine agonist efficacy: the concentration-dependent association rate of the G protein to the agonist-bound receptor, and the catalytic rate of G protein activation by the receptor. While both rate constants scale proportionally with reported molecular efficacy metrics, we find that the concentration-dependent association rate is the governing parameter across multiple distinct GPCR-ligand pairings. Our model explains discrepancies in signaling outcomes observed between physiological observations and cellular model systems that rely on receptor overexpression.

Identifying target-specific drugs remains a challenge in pharmacology, especially for highly homologous proteins such as dopamine receptors D2R and D3R. Differences in target-specific cryptic druggable sites for such receptors arise from the distinct conformational ensembles underlying their dynamic behavior. While Molecular Dynamics (MD) simulations has emerged as a powerful tool for dissecting protein dynamics, the sheer volume of MD data requires scalable and unbiased data analysis strategies to pinpoint residue communities regulating conformational state ensembles. We present the Dynamically Resolved Universal Model for BayEsiAn network Tracking (DRUMBEAT) interpretable machine learning algorithm and validate it by identifying residue communities that enable the deactivation of the β2-adrenergic receptor. Further, upon analyzing dopamine receptor dynamics we identify distinct and non-conserved residue communities around the contacts F1704.62_F172ECL2 and S1464.38_G14134.56 that are specific to D3R conformational transitions compared to D2R. This information can be tapped to design subtype-specific drugs for neuropsychiatric and substance use disorders.

The folded structure of a protein is understood to be an optimal energy state. However, previous studies have shown that certain amino acid residue positions that play a critical role in protein function are often in a suboptimal energy state or “frustrated”. Here, we leverage over 1200 three-dimensional structures of G protein-coupled receptors (GPCRs) to demonstrate that residues at the interface between GPCR and its ligand or G protein contain a higher density of frustrated residues compared to other structural regions in the receptor. Likewise, the Gα subunit of the trimeric G proteins shows multiple clusters of highly frustrated residues on its surface that overlap with their effector protein (Gβγ, RGS, Adenyl cyclase, Ric8) binding interfaces. Compared to the co-evolved GPCR:G protein complexes, engineered protein complexes, such as those facilitated by molecular degraders, show a much greater density of highly frustrated residues in the degrader interface. Our study highlights the use of protein frustration as an invaluable tool to evaluate both native protein-protein interfaces and design strategies to facilitate engineered protein complexes.

Glucose-dependent insulinotropic peptide receptor (GIPR) stimulates insulin release and regulates metabolic homeostasis. GIPR function is shaped by spatiotemporal trafficking of this G protein-coupled receptor (GPCR). While GPCR endocytosis is traditionally associated with β-arrestin, GIPR internalization is only modestly dependent on this pathway. In this study, we demonstrate that GIPR engages a cytoskeletal motor, myosin VI to drive receptor endocytosis. GIPR engages the adaptor-motor complex through a PDZ-binding motif (PBM) at its C-ail. Interestingly, β-arrestin binding to phosphorylated residues upstream of the PBM enhance myosin VI recruitment and activation. GIPR internalization is dependent on both receptor phosphorylation and the PBM site to recruit β-arrestin and myosin VI, respectively. Synergistic engagement of β-arrestin and myosin VI results in desensitization of GIP-stimulated cAMP signaling while activating pERK1/2 from endosomal compartments. Blocking myosin VI activity enhances insulin release in pancreatic beta cells, demonstrating a novel role for this pathway in regulating the physiological effects of GIPR. Our findings highlight the direct convergence of two independent trafficking pathways at the level of the receptor C-tail, with implications for the nuanced regulation of individual GPCRs through the differential engagement of β-arrestin and myosin VI. Significance:GIPR has emerged as a frontline drug target in type 2 diabetes and obesity. Cellular effects of GIPR are regulated by receptor internalization and desensitization through mechanisms that are unclear. Here, we identify a novel GIPR trafficking pathway through the engagement of a cytoskeletal motor, myosin VI. Myosin VI and β-arrestin synergistically regulate GIPR endocytosis, signaling and insulin response in pancreatic beta cells. Our study highlights the convergence of two parallel trafficking mechanisms in GPCR function with potential implications in targeting metabolic disorders.

The intracellular loops of G protein -coupled receptors (GPCRs) have been shown to play a key role in G protein coupling and selectivity. We recently showed that the intrinsically disordered third intracellular loop (ICL3) of 0 2-adrenergic receptor is dynamic and equilibrates between open and closed conformations to regulate the G protein coupling. In this study, using the extensive molecular dynamics simulations in multi -lipid bilayer models, we show that the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) stabilizes the active state of 0 2-adrenergic receptor by keeping ICL3 in an open conformation. This stabilization results in a tilt of the receptor within the membrane. Additionally, the ganglioside lipid, GM3 interacts with extracellular loops, impacting the ligand binding site allosterically. This demonstrates the active role of the chemistry of lipids in stabilizing specific GPCR confor

G protein-coupled receptor (GPCR) endocytosis is canonically associated with beta-arrestins. Here, we delineate a beta-arrestin-independent endocytic pathway driven by the cytoskeletal motor, myosin VI. Myosin VI engages GIPC, an adaptor protein that binds a PDZ sequence motif present at the C-terminus of several GPCRs. Using the D2 dopamine receptor (D2R) as a prototype, we find that myosin VI regulates receptor endocytosis, spatiotemporal localization, and signaling. We find that access to the D2R C-tail for myosin VI-driven internalization is controlled by an interaction between the C-tail and the third intracellular loop of the receptor. Agonist efficacy, co-factors, and GIPC expression modulate this interaction to tune agonist trafficking. Myosin VI is differentially regulated by distinct GPCR C-tails, suggesting a mechanism to shape spatiotemporal signaling profiles in different ligand and physiological contexts. Our biophysical and structural insights may advance orthogonal therapeutic strategies for targeting GPCRs through cytoskeletal motor proteins.

Cardiac myosin binding protein C (cMyBP-C) is exclusively found in the C-zone of the heart sarcomere where it associates with myosin and actin to modulate heart contractility. About 40% of genetic cardiomyopathy cases are linked to defects in cMyBP-C. Studying this protein in its native configuration is challenging as it is part of a multi-protein sarcomeric complex, which together regulate β-cardiac myosin function. One promising way to circumvent this problem is to mimic the myosin thick filament with DNA nanostructures where β-cardiac myosin and cMyBP-C can be spatially related in a precise way so that their interaction with actin can be studied without other confounding factors.

Dilated cardiomyopathy (DCM) is a condition characterized by impaired cardiac function, due to myocardial hypo-contractility, and is associated with point mutations in β-cardiac myosin, the molecular motor that powers cardiac contraction. Myocardial function can be modulated through sequestration of myosin motors into an auto-inhibited “super-relaxed” state (SRX), which may be further stabilized by a structural state known as the “interacting heads motif” (IHM). Here, we sought to determine whether hypo-contractility of DCM myocardium results from reduced function of individual myosin molecules or from decreased myosin availability to interact with actin due to increased IHM/SRX stabilization. We used an established DCM myosin mutation, E525K, and characterized the biochemical and mechanical activity of wild-type and mutant human β-cardiac myosin constructs that differed in the length of their coiled-coil tail, which dictates their ability to form the IHM/SRX state. We found that short-tailed myosin constructs exhibited low IHM/SRX content, elevated actin-activated ATPase activity, and fast velocities in unloaded motility assays. Conversely, longer-tailed constructs exhibited higher IHM/SRX content and reduced actomyosin ATPase and velocity. Our modeling suggests that reduced velocities may be attributed to IHM/SRX-dependent sequestration of myosin heads. Interestingly, longer-tailed E525K mutants showed no apparent impact on velocity or actomyosin ATPase at low ionic strength but stabilized IHM/SRX state at higher ionic strength. Therefore, the hypo-contractility observed in DCM may be attributable to reduced myosin head availability caused by enhanced IHM/SRX stability in E525K mutants.

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 β2-adrenergic receptor (β2AR). 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 β2AR 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.

The structural basis of the slow ATP turnover in the super-relaxed (SRX) state of cardiac myosin is currently unclear. There is evidence that a conformation with the heads folded back on the tail and interacting with each other called the interacting heads motif (IHM) can stabilize the SRX biochemical state. We designed a FRET biosensor of the IHM structural state, by utilizing a beta-cardiac myosin heavy meromyosin construct with a C-terminal GFP tag (M2β HMM). We observed FRET between the C-terminal GFP tag and Cy3ATP in the active site, that we predicted would only occur with the heads folded back in the IHM.

Throughout the cell, motor proteins work together to drive numerous molecular processes and functions. For example, ensembles of myosin motors collectively transport vesicles and organelles, maintain membrane homeostasis, and drive muscle contraction. Studying these motors in groups has become increasingly important with work demonstrating the emergence of ensemble behavior distinct from individual motor behavior. One powerful technique that has been used in the last decade is DNA nanotechnology, which provides precise control over spacing and organization of patterned motor proteins. Until recently, however, most studies combining DNA nanostructures and molecular motors have been confined to discrete DNA structures with limited attachment points for motor proteins. In this chapter, we describe a new approach for making synthetic motor filaments using DNA nanotubes. We present methods for preparing myosin VI-labeled nanotubes and testing these nanotubes using a general in vitro motility setup. Overall, these nanotubes can easily be used to study other large ensembles of molecular motors, such as muscle myosin or ciliary dynein, both proteins that work in large motor ensembles to drive key cellular functions.

Supplementary Figure S6. Validating immunodepletion of NK cells and basophils in RAG1-/- mice.

Supplementary Figure S8. Splenic-derived RAG1-/- NK cell purity assessment.

Supplementary Figure S3. Validation of GL26-Cit cells independently transduced with gal-1-specific or control shRNA.

Supplementary Figure S4. GL26-Cit-gal1i cells do not undergo spontaneous cell-death in-vitro.