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.

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 S1. Plasmid map of the pCI-neo-mCitrine expression vector.

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.

Legend for Supplementary Figures S1-S8.

Description of additional methods and procedures used in the study.

Supplementary Figure S5. Morphometric analysis of 48hr post-implantation intracranial GL26-Cit gliomas (shown in Figure 1G).

The third intracellular loop (ICL3) of the G protein-coupled receptor (GPCR) fold is important for the signal transduction process downstream of receptor activation 1 – 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 signalling 4 . Previous studies focusing on the β 2 adrenergic receptor (β 2 AR) suggest that ICL3 is involved in the structural process of receptor activation and signalling 5 – 7 . Here we derive mechanistic insights into the role of ICL3 in β 2 AR 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.

Supplementary Figure S7. Anti-CD3ε immunolabeled brain tissue sections from mouse A1 (top image) and mouse N2 (bottom image) shown in Figure 5I.

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose over-expression with this motor in cancer enhances cell migration. Using a range of biophysical techniques, including motility assays, FRET-based conformational sensors, optical trapping, and DNA origami-based cargo scaffolds to probe the individ-ual and ensemble properties of GIPC-myosin VI motility, we report that the GIPC myosin-interacting region (MIR) releases an autoinhibitory interaction within myosin VI. We show that the resulting conformational changes in the myosin lever arm, including the proximal tail domain, increase the flexibility of the adaptor-motor linkage, and that increased flexibility cor-relates with faster actomyosin association and dissociation rates. Taken together, the GIPC MIR-myosin VI interaction stimulates a twofold to threefold increase in ensemble cargo speed. Furthermore, the GIPC MIR-myosin VI ensembles yield similar cargo run lengths as forced processive myosin VI di-mers. We conclude that the emergent behavior from these individual aspects of myosin regulation is the fast, processive, and smooth cargo transport on cellular actin networks. Our study delineates the multimodal regulation of myosin VI by the cargo adaptor GIPC, while highlighting linkage flexibility as a novel biophysical mechanism for modulating cellular cargo motility.

Endocytic recycling is a complex itinerary, critical for many cellular processes. Membrane tubulation is a hallmark of recycling endosomes (REs), mediated by KIF13A, a kinesin-3 family motor. Understanding the regulatory mechanism of KIF13A in RE tubulation and cargo recycling is of fundamental importance but is overlooked. Here, we report a unique mechanism of KIF13A dimerization modulated by Rab22A, a small guanosine triphosphatase, during RE tubulation. A conserved proline between neck coil-coiled-coil (NC-CC1) domains of KIF13A creates steric hindrance, rendering the motors as inactive monomers. Rab22A plays an unusual role by binding to NC-CC1 domains of KIF13A, relieving proline-mediated inhibition and facilitating motor dimerization. As a result, KIF13A motors produce balanced motility and force against multiple dyneins in a molecular tug-of-war to regulate RE tubulation and homeostasis. Together, our findings demonstrate that KIF13A motors are tuned at a single-molecule level to function as weak dimers on the cellular cargo.

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s-1) of the Dab2 MIR-myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 μM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and "stop-and-go" motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR-myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor-cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

The eukaryotic kinase domain has multiple intrinsically disordered regions whose conformation dictates kinase activity. Small molecule kinase inhibitors (SMKIs) rely on disrupting the active conformations of these disordered regions to inactivate the kinase. While SMKIs are selected for their ability to cause this disruption, the allosteric effects of conformational changes in disordered regions is limited by a lack of dynamic information provided by traditional structural techniques. In this study, we integrated multiscale molecular dynamics simulations with FRET sensors to characterize a novel allosteric mechanism that is selectively triggered by SMKI binding to the protein kinase Cα domain. The indole maleimide inhibitors BimI and sotrastaurin were found to displace the Gly-rich loop (G-loop) that normally shields the ATP-binding site. Displacement of the G-loop interferes with a newly identified, structurally conserved binding pocket for the C1a domain on the N lobe of the kinase domain. This binding pocket, in conjunction with the N-terminal regulatory sequence, masks a diacylglycerol (DAG) binding site on the C1a domain. SMKI-mediated displacement of the G-loop released C1a and exposed the DAG binding site, enhancing protein kinase Cα translocation both to synthetic lipid bilayers and to live cell membranes in the presence of DAG. Inhibitor chemotype determined the extent of the observed allosteric effects on the kinase domain and correlated with the extent of membrane recruitment. Our findings demonstrate the allosteric effects of SMKIs beyond the confines of kinase catalytic conformation and provide an integrated computational-experimental paradigm to investigate parallel mechanisms in other kinases.

Significance An outstanding challenge in GPCR pharmacology is quantifying the system-independent functional effects of ligand-receptor interactions. Current efforts to measure ligand efficacy at the level of the receptor (i.e., molecular efficacy) are limited by the requirement of extensive purification of receptor and G proteins to homogeneity. In this study, we present an accessible, scalable technology for the single point measurement of the molecular efficacy of GPCR ligands. Integrating this technology with insights from molecular dynamics simulations, we reveal that the transition of the G protein from an intermediate to a fully coupled interaction with the GPCR is a structural determinant of ligand molecular efficacy.

ER/K α-helices are a subset of single alpha helical domains, which exhibit unusual stability as isolated protein secondary structures. They adopt an elongated structural conformation, while regulating the frequency of interactions between proteins or polypeptides fused to their ends. Here we review recent advances on the structure, stability and function of ER/K α-helices as linkers (ER/K linkers) in native proteins. We describe methodological considerations in the molecular cloning, protein expression and measurement of interaction strengths, using sensors incorporating ER/K linkers. We highlight biological insights obtained over the last decade by leveraging distinct biophysical features of ER/K-linked sensors. We conclude with the outlook for the use of ER/K linkers in the selective modulation of dynamic cellular interactions.

Despite the crowded nature of the cellular milieu, ligand–GPCR–G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor–effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR–G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the β 2 -adrenergic receptor (β 2 -AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. β 2 -AR–Gα C termini interactions alter receptor conformation, which persists for ~90 s following Gα C terminus dissociation. Non-cognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface.

While unconventional myosins interact with different stages of the endocytic pathway, they are ascribed a transport function that is secondary to the protein complexes that control organelle identity. Endosomes are subject to a dynamic, continuous flux of proteins that control their characteristic properties, including their motility within the cell. Efforts to describe the changes in identity of this compartment have largely focused on the adaptors present on the compartment and not on the motile properties of the compartment itself. In this study, we use a combination of optogenetic and chemical‐dimerization strategies to target exogenous myosin VI to early endosomes, and probe its influence on organelle motility, morphology and identity. Our analysis across timescales suggests a model wherein the artificial engagement of myosin VI motility on early endosomes restricts microtubule‐based motion, followed by morphological changes characterized by the rapid condensation and disintegration of organelles, ultimately leading to the enhanced overlap of markers that demarcate endosomal compartments. Together, our findings show that synthetic engagement of myosin VI motility is sufficient to alter organelle homeostasis in the endocytic pathway.