Rajesh K. Murarka

β-Arrestins (βarr1 and βarr2) are key transducers of G protein-coupled receptor (GPCR) signaling, orchestrating both shared and isoform-specific intracellular pathways. Phosphorylation of the receptor C-terminal tail by GPCR kinases encodes regulatory "barcodes" that modulate β-arrestin conformations and interactions with downstream effectors. However, how distinct phosphorylation patterns shape β-arrestin structure and function remains poorly understood. In this study, we integrate all-atom molecular dynamics simulations with machine learning, including graph neural networks, to systematically characterize the barcode-specific conformational landscape of β-arrestins bound to the phosphorylated vasopressin receptor 2 tail (V2Rpp). We find that V2Rpp engages βarr1 more stably than βarr2, mediated by isoform-specific residue contacts that trigger distinct allosteric responses. These include differential interdomain rotations and rearrangements in key structural motifs, potentially facilitating selective effector protein engagement. Furthermore, we identify critical residue networks that transmit phosphorylation signals to effector-binding interfaces in a barcode- and isoform-specific manner. Notably, βarr1 exhibits stronger allosteric coupling between V2Rpp and c-edge loop 2 compared to βarr2, which is consistent with its enhanced membrane association. Together, these findings advance our understanding of the molecular mechanisms by which β-arrestins interpret GPCR phosphorylation signatures, offering a framework that could aid in the design of pathway-selective therapeutics.

β -arrestins ( β arr1 and β arr2) are key transducers of G protein-coupled receptor (GPCR) signaling, orchestrating both shared and isoform-specific intracellular pathways. Phosphorylation of the receptor C-terminal tail by GPCR kinases encodes regulatory “barcodes” that modulate β -arrestin conformations and interactions with downstream effectors. However, how distinct phosphorylation patterns shape β -arrestin structure and function remains poorly understood. Here, we combine all-atom molecular dynamics simulations with machine learning and graph neural networks to systematically map the barcode-dependent conformational landscapes of β -arrestins bound to the phosphorylated vasopressin receptor 2 tail (V2Rpp). We find that V2Rpp engages β arr1 more stably than β arr2, mediated by isoform-specific residue contacts that trigger distinct allosteric responses. These include differential interdomain rotations and rearrangements in key structural motifs, potentially facilitating selective effector protein engagement. Furthermore, we identify critical residue networks that transmit phosphorylation signals to effector-binding interfaces in a barcode- and isoform-specific manner. Notably, β arr1 exhibits stronger allosteric coupling between V2Rpp and c-edge loop 2 compared to β arr2, which is consistent with its enhanced membrane association. Together, these findings advance our understanding of the molecular mechanisms by which β -arrestins interpret GPCR phosphorylation signatures, offering a framework that could aid in designing pathway-selective therapeutics.

Activation of heterotrimeric G-proteins (G αβγ ) downstream to receptor tyrosine kinases (RTKs) is a well-established crosstalk between the signaling pathways mediated by G-protein coupled receptors (GPCRs) and RTKs. While GPCR serves as a guanine exchange factor (GEF) in the canonical activation of G α that facilitates the exchange of GDP for GTP, the mechanism through which RTK phosphorylations induce G α activation remains unclear. Recent experimental studies revealed that the epidermal growth factor receptor (EGFR), a well-known RTK, phosphorylates the helical domain tyrosine residues Y154 and Y155 and accelerates the GDP release from the G α i3, a subtype of G α -protein. Using well-tempered metadynamics and extensive unbiased molecular dynamics simulations, we captured the GDP release event and identified the intermediates between bound and unbound states through Markov state models. The additional negative charges introduced by phosphorylations rewired the inter-residue interactions and significantly weakened the salt bridges at the domain interface, contributing to the increased separation of the Ras-like and helical domains of G-protein. Furthermore, the unfolding of helix α F resulted in greater flexibility near the hinge region, facilitating a greater distance between domains in the phosphorylated G α i3. The release of GDP in the phosphorylated G-protein occurred at a faster rate compared to the unphosphorylated state, caused by increased fluctuations in conserved regions of P-loop, switch 1, and switch 2. Overall, this study provides atomistic insights into the activation of G-proteins induced by RTK phosphorylations and identifies the specific structural motifs involved in the process. The knowledge gained from the study could establish a foundation for targeting non-canonical signaling pathways and developing therapeutic strategies against the ailments associated with dysregulated G-protein signaling.

The molecular basis of receptor bias in G protein-coupled receptors (GPCRs) caused by mutations that preferentially activate specific intracellular transducers over others remains poorly understood. Two experimentally identified biased variants of β ₂ -adrenergic receptors ( β ₂ AR), a prototypical GPCR, are a triple mutant (T68F, Y132A, and Y219A) and a single mutant (Y219A); the former bias the receptor towards the β -arrestin pathway by disfavoring G protein engagement, while the latter induces G protein signaling explicitly due to selection against GPCR kinases (GRKs) that phosphorylate the receptor as a prerequisite of β -arrestin binding. Though rigorous characterizations have revealed functional implications of these mutations, the atomistic origin of the observed transducer selectivity is not clear. In this study, we investigate the allosteric mechanism of receptor bias in β ₂ AR using microseconds of all-atom Gaussian accelerated molecular dynamics (GaMD) simulations. Our observations reveal distinct rearrangements in transmembrane helices, intracellular loop 3, and critical residues R131 ^3.50 and Y326 ^7.53 in the conserved motifs D(E)RY and NPxxY for the mutant receptors, leading to their specific transducer interactions. The reorganization of allosteric communications from the extracellular agonist BI-167107 to the intracellular receptor-transducer interfaces drives the conformational rearrangements responsible for receptor bias in the single and triple mutants. The molecular insights into receptor bias of β ₂ AR presented here could improve the understanding of biased signaling in GPCRs, potentially opening new avenues for designing novel therapeutics with fewer side effects and superior efficacy.

The large conformational flexibility of G protein-coupled receptors (GPCRs) has been a puzzle in structural and pharmacological studies for the past few decades. Apart from structural rearrangements induced by ligands, enzymatic phosphorylations by GPCR kinases (GRKs) at the carboxy-terminal tail (C-tail) of a GPCR also make conformational alterations to the transmembrane helices and facilitates the binding of one of its transducer proteins named β-arrestin. The phosphorylation-induced conformational transition of the receptor that causes specific binding to β-arrestin but prevents the association of other transducers such as G proteins lacks atomistic understanding and is elusive to experimental studies. Using microseconds of all-atom conventional and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigate the allosteric mechanism of phosphorylation induced-conformational changes in β₂-adrenergic receptor, a well-characterized GPCR model system. Free energy profiles reveal that the phosphorylated receptor samples a new conformational state in addition to the canonical active state corroborating with recent nuclear magnetic resonance experimental findings. The new state has a smaller intracellular cavity that is likely to accommodate β-arrestin better than G protein. Using contact map and inter-residue interaction energy calculations, we found the phosphorylated C-tail adheres to the cytosolic surface of the transmembrane domain of the receptor. Transfer entropy calculations show that the C-tail residues drive the correlated motions of TM residues, and the allosteric signal is relayed via several residues at the cytosolic surface. Our results also illustrate how the redistribution of inter-residue nonbonding interaction couples with the allosteric communication from the phosphorylated C-tail to the transmembrane. Atomistic insight into phosphorylation-induced β-arrestin specific conformation is therapeutically important to design drugs with higher efficacy and fewer side effects. Our results, therefore, open novel opportunities to fine-tune β-arrestin bias in GPCR signaling.

The large conformational flexibility of G protein-coupled receptors (GPCRs) has been a puzzle in structural and pharmacological studies for the past few decades. Apart from structural rearrangements induced by ligands, enzymatic phosphorylations by GPCR kinases (GRKs) at the carboxy-terminal tail (C-tail) of a GPCR also makes conformational alterations to the transmembrane helices and facilitates the binding of one of its transducer proteins named β-arrestin. Phosphorylation-induced conformational transition of the receptor that causes specific binding to β-arrestin but prevents the association of other transducers such as G proteins lacks atomistic understanding and is elusive to experimental studies. Using microseconds of all-atom conventional and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigate the allosteric mechanism of phosphorylation induced-conformational changes in β ₂ -adrenergic receptor, a well-characterized GPCR model system. Free energy profiles reveal that the phosphorylated receptor samples a new conformational state in addition to the canonical active state corroborating with recent nuclear magnetic resonance experimental findings. The new state has a smaller intracellular cavity that is likely to accommodate β-arrestin better than G protein. Using contact map and inter-residue interaction energy calculations, we found the phosphorylated C-tail adheres to the cytosolic surface of the transmembrane domain of the receptor. Transfer entropy calculations show that the C-tail residues drive the correlated motions of TM residues, and the allosteric signal is relayed via several residues at the cytosolic surface. Our results also illustrate how the redistribution of inter-residue nonbonding interaction couples with the allosteric communication from the phosphorylated C-tail to the transmembrane. Atomistic insight into phosphorylation-induced β-arrestin specific conformation is therapeutically important to design drugs with higher efficacy and fewer side effects. Our results therefore open novel opportunities to fine-tune β-arrestin bias in GPCR signaling.

Molecular dynamics with coarse-grained models is nowadays extensively used to simulate biomolecular systems at large time and size scales, compared to those accessible to all-atom molecular dynamics. In this review article, we describe the physical basis of coarse-grained molecular dynamics, the coarse-grained force fields, the equations of motion and the respective numerical integration algorithms, and selected practical applications of coarse-grained molecular dynamics. We demonstrate that the motion of coarse-grained sites is governed by the potential of mean force and the friction and stochastic forces, resulting from integrating out the secondary degrees of freedom. Consequently, Langevin dynamics is a natural means of describing the motion of a system at the coarse-grained level and the potential of mean force is the physical basis of the coarse-grained force fields. Moreover, the choice of coarse-grained variables and the fact that coarse-grained sites often do not have spherical symmetry implies a non-diagonal inertia tensor. We describe selected coarse-grained models used in molecular dynamics simulations, including the most popular MARTINI model developed by Marrink’s group and the UNICORN model of biological macromolecules developed in our laboratory. We conclude by discussing examples of the application of coarse-grained molecular dynamics to study biologically important processes.

In order to understand the role of osmolytes in regulating physicochemical behavior of proteins, we investigated the influence of protein destabilizing (urea and guanidinium chloride) and stabilizing osmolytes (TMAO, glycerol, and betaine) on a model salt-bridge (SB) formed between structural analogues of arginine and glutamate/aspartate sidechains in a solvent continuum using first-principles quantum chemical calculations based on DFT and MP2 methods. The binding strength of the osmolyte with the SB is found to be in the order of betaine > TMAO > Gdm⁺ > glycerol > urea. The osmolytes (TMAO and betaine) that preferentially bind to the SB cation have a marginal influence on SB stability. Also, pure π–π stacking interaction between Gdm⁺ and the SB cation plays an insignificant role in destabilizing the SB. In fact, the interaction strength of osmolytes with the SB anion mainly determines the stability of SB. For instance, a competition between Gdm⁺ and the SB cation to bind with the SB anion is responsible for instability and subsequent dissociation of the SB. The competition provided by other osmolytes is too weak to break the SB. Exploiting this information, we designed three structural derivatives of Gdm⁺, all having a stronger interaction with SB anion, and thereby show a stronger SB dissociation potential. Furthermore, we find an excellent linear anti-correlation between SB interaction energy and the energy of interaction between osmolyte and the SB anion, which suggests that by knowing only the strength of osmolyte⋯acetate interaction, one can predict the influence of osmolytes on the salt-bridge instability. This information is useful in fine-tuning the SB dissociation power of Gdm⁺, which has a practical significance in obtaining the mechanistic insight into the influence of GdmCl on protein stability. Our results also provide a basis for understanding the chemistry of other ion-pairs formed between a cationic hydrogen donor and an anionic acceptor.

There is ample evidence that both native functions and pathogenic aggregation of α-synuclein are intimately dependent on lipid interactions and fatty acid type; the regulatory mechanism however remains unclear. In the present work, using extensive atomistic molecular dynamics simulations and enhanced-sampling, we have focused on exploring the mechanism of fatty acid dependent regulation of monomeric α-Syn₁₀₀ in a native synaptic vesicle-like membrane. Our results show that α-Syn₁₀₀ spontaneously binds to the membrane through its N-terminal region (residues 1–34), where the depth of membrane insertion, the structure, and orientation of the membrane-bound α-Syn₁₀₀ and its impact on membrane structure are modulated by docosahexaenoic acid (DHA). DHA is a polyunsaturated fatty acid abundantly found in the brain and known to promote the oligomerization of α-synuclein. We found that DHA exhibits marked propensity to interact with monomeric α-Syn₁₀₀ and modulates the microenvironment of the protein by preferentially sorting DHA-containing phospholipids, depleting other phospholipids and cholesterol as well as increasing the proportion of anionic to neutral lipids in the immediate vicinity of the protein. Owing to the unique conformational flexibility, DHA chains form more lipid-packing defects in the membrane and efficiently coat the membrane-embedded surface of the protein, compared to the saturated and monounsaturated fatty acids. DHA thus makes the bilayer more amiable to protein adsorption and less prone to α-synuclein-induced perturbation associated with cytotoxicity. Indeed, in the absence of DHA, we observed significant thinning of the local bilayer membrane induced by α-Syn₁₀₀. Though α-Syn₁₀₀ is predominantly α-helical in membranes studied here, in the presence of DHA we observe formation of β-sheet/β-strands in the C-terminal region (residues 35–100) of α-Syn₁₀₀, which is extended out from the membrane surface. Notably, DHA induces β structure in the NAC domain of α-Syn₁₀₀ and promotes extended conformations as well as large solvent exposure of this hydrophobic domain, properties that are known to facilitate self-assembly of α-synuclein. To the best of our knowledge, this study for the first time provides the atomistic insights into DHA-induced regulatory mechanism of monomeric α-synuclein, having implications in protein structure and its physiological/pathological functions.