Mechanism Of Allosteric Communication Research Articles

Mechanism of allosteric communication in the neuromuscular acetylcholine receptors

Mechanism of allosteric communication in the neuromuscular acetylcholine receptors

Probing conformational landscapes and mechanisms of allosteric communication in the functional states of the ABL kinase domain using multiscale simulations and network-based mutational profiling of allosteric residue potentials.

In the current study, multiscale simulation approaches and dynamic network methods are employed to examine the dynamic and energetic details of conformational landscapes and allosteric interactions in the ABL kinase domain that determine the kinase functions. Using a plethora of synergistic computational approaches, we elucidate how conformational transitions between the active and inactive ABL states can employ allosteric regulatory switches to modulate intramolecular communication networks between the ATP site, the substrate binding region, and the allosteric binding pocket. A perturbation-based network approach that implements mutational profiling of allosteric residue propensities and communications in the ABL states is proposed. Consistent with biophysical experiments, the results reveal functionally significant shifts of the allosteric interaction networks in which preferential communication paths between the ATP binding site and substrate regions in the active ABL state become suppressed in the closed inactive ABL form, which in turn features favorable allosteric coupling between the ATP site and the allosteric binding pocket. By integrating the results of atomistic simulations with dimensionality reduction methods and Markov state models, we analyze the mechanistic role of macrostates and characterize kinetic transitions between the ABL conformational states. Using network-based mutational scanning of allosteric residue propensities, this study provides a comprehensive computational analysis of long-range communications in the ABL kinase domain and identifies conserved regulatory hotspots that modulate kinase activity and allosteric crosstalk between the allosteric pocket, ATP binding site, and substrate binding regions.

The Role of Conformational Dynamics and Allostery in the Control of Distinct Efficacies of Agonists to the Glucocorticoid Receptor.

Glucocorticoid receptor (GR) regulates various cellular functions. Given its broad influence on metabolic activities, it has been the target of drug discovery for decades. However, how drugs induce conformational changes in GR has remained elusive. Herein, we used five GR agonists (dex, AZ938, pred, cor, and dibC) with different efficacies to investigate which aspect of the ligand induced the differences in efficacy. We performed molecular dynamics simulations on the five systems (dex-, AZ938-, pred-, cor-, and dibC-bound systems) and observed a distinct discrepancy in the conformation of the cofactor TIF2. Moreover, we discovered ligand-induced differences regarding the level of conformational changes posed by the binding of cofactor TIF2 and identified a pair of essential residues D590 and T39. We further found a positive correlation between the efficacies of ligands and the interaction of the two binding pockets’ domains, where D590 and T739 were involved, implying their significance in the participation of allosteric communication. Using community network analysis, two essential communities containing D590 and T739 were identified with their connectivity correlating to the efficacy of ligands. The potential communication pathways between these two residues were revealed. These results revealed the underlying mechanism of allosteric communication between the ligand-binding and cofactor-binding pockets and identified a pair of important residues in the allosteric communication pathway, which can serve as a guide for future drug discovery.

An investigation into the allosteric mechanism of GPCR A2A adenosine receptor with trajectory-based information theory and complex network model

G protein-coupled receptors (GPCRs), a large superfamily of transmembrane (TM) proteins, allosterically transduce the signal of ligand binding in the extracellular (EC) domain to couple to effector proteins in the intracellular (IC) domain, therefore forming the largest class of drug targets. The A2A adenosine receptor (A2AAR), a class-A GPCR, has been extensively studied as it offers numerous possibilities for therapeutic applications. However, the mechanism of allosteric communication between EC and IC domains is not completely clear. In this work, we utilize torsional mutual information to quantify the correlated motions of residue pairs from its molecular dynamics (MD) simulation trajectories, and further use the complex network model to obtain allosteric pipelines and hubs. The identified allosteric communication pipelines mainly transmit the signal from EC domain to the cytoplasmic ends of TM helix 5 (TM5), TM6 and TM7. The allosteric hubs, mostly located at TM5, TM6 and TM7, play an important role in mediating allosteric signal transmission to keep the receptor rigid and prevent G protein from binding to IC domain, which can explain the reason why their mutations distant from ligand-binding site do not affect the ligand binding affinity but affect the ligand efficacy. Additionally, we identify the key residues located in antagonist ZM241385 binding pocket which mediate multiple allosteric pathways and have been experimentally proven to play a critical role in affecting the ligand potency. This study is helpful for understanding the allosteric communication mechanism of A2AAR, and can provide valuable information for the structure-based drug design of GPCRs. Communicated by Ramaswamy H. Sarma

Identifying Functional Hotspot Residues for Biased Ligand Design in G-Protein-Coupled Receptors.

G-protein-coupled receptors (GPCRs) mediate multiple signaling pathways in the cell, depending on the agonist that activates the receptor and multiple cellular factors. Agonists that show higher potency to specific signaling pathways over others are known as "biased agonists" and have been shown to have better therapeutic index. Although biased agonists are desirable, their design poses several challenges to date. The number of assays to identify biased agonists seems expensive and tedious. Therefore, computational methods that can reliably calculate the possible bias of various ligands ahead of experiments and provide guidance, will be both cost and time effective. In this work, using the mechanism of allosteric communication from the extracellular region to the intracellular transducer protein coupling region in GPCRs, we have developed a computational method to calculate ligand bias ahead of experiments. We have validated the method for several β-arrestin-biased agonists in β2-adrenergic receptor (β2AR), serotonin receptors 5-HT1B and 5-HT2B and for G-protein-biased agonists in the κ-opioid receptor. Using this computational method, we also performed a blind prediction followed by experimental testing and showed that the agonist carmoterol is β-arrestin-biased in β2AR. Additionally, we have identified amino acid residues in the biased agonist binding site in both β2AR and κ-opioid receptors that are involved in potentiating the ligand bias. We call these residues functional hotspots, and they can be used to derive pharmacophores to design biased agonists in GPCRs.

Mechanism of Allosteric Communication in GPCR Activation from Microsecond Scale Molecular Dynamics Simulations

G protein coupled receptors (GPCRs) are membrane proteins that signal through effector proteins bound at the intracellular (IC) interface. Under physiological conditions, GPCRs adopt multiple inactive and active conformations, thereby signaling through diverse signaling pathways. Binding of agonists at the extracellular (EC) domain initiates a conformational change in the receptor leading to activation. Antagonists and inverse agonists suppress activation by stabilizing one of the inactive states. The communication between the agonist/antagonist binding site and the IC interface takes place through allosteric coupling of distant receptor domains. However, the detailed mechanism of allosteric communication in GPCR signaling is not well understood. Using ensembles of molecular dynamics (MD) simulations on eight class A GPCRs, we showed that allosteric communication between the antagonist binding site and the IC interface stabilizes the inactive state, and disruption of this allosteric communication due to agonist binding leads to activation. Furthermore, the fundamental framework and mechanism of allosteric communication are conserved among multiple GPCRs, as is evident from the conserved allosteric hubs (residues that mediate multiple allosteric pathways) in these receptors. Mutating the allosteric hubs either suppress activation or impart constitutive activity, suggesting the key role of these residues in GPCR function. We have shown that, besides stabilizing GPCR functional states, allosteric communication is also responsible for transitions among functional states. Analysis of the deactivation dynamics of two class A GPCRs (β2 adrenergic receptor and neurotensin receptor 1) shows that several allosteric hubs in the middle of the transmembrane domain undergo concerted dihedral changes during the transition from the active to the inactive state. These observations provide valuable insights into the complex mechanism of GPCR activation and membrane protein function in general.

Mapping Allosteric Communication Pipelines in GPCRs from Microsecond Timescale Molecular Dynamics Simulations

G protein coupled receptors (GPCRs) are 7 transmembrane (TM) helical proteins that signal through effector proteins bound at the intracellular (IC) interface. Agonist binding at the extracellular (EC) domain triggers conformational change at the IC domain leading to receptor activation. However, the mechanism of allosteric communication and the amino acids involved in the pipelines of allosteric communication (allosteric pathways) are not known yet. In this study, we have mapped the allosteric pipelines of communication in β2-adrenergic receptor (β2AR) using microseconds timescale Molecular Dynamics trajectories. Using mutual information in the internal coordinates, we have compared the allosteric pipelines among three β2AR conformations; inverse agonist bound inactive state, agonist bound intermediate state, and agonist and G protein bound active state. Strong allosteric communication along the TM domains stabilize the receptor in the inactive and active conformations. However the agonist bound intermediate state is dynamic showing weakened communication between the EC and IC domains. We have identified the residues that mediate multiple allosteric pipelines as allosteric hubs. These allosteric hubs have also been identified with the mutations that alter the receptor efficacy without altering the ligand binding. The role of such mutations were not previously understood.We have identified the residue networks involved in a continuous communication pipeline from the EC loops to the G protein coupling site. One of the termination points of the activation signal is the engineered Zn2+ binding site at the IC interface, which is a known positive allosteric modulator binding site in β2AR. We have shown how our method in conjunction with MD simulations can identify allosteric pockets which can then be used for screening allosteric drugs in GPCRs.

Switch II Mutants Reveal Coupling between the Nucleotide- and Actin-Binding Regions in Myosin V

Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region.