The allosteric communication between the agonist binding site and the G protein or β-arrestin coupling sites in G protein-coupled receptors (GPCRs) play an important role in determining ligand efficacy towards these two signaling pathways... more
The allosteric communication between the agonist binding site and the G protein or β-arrestin coupling sites in G protein-coupled receptors (GPCRs) play an important role in determining ligand efficacy towards these two signaling pathways and hence the ligand bias. Knowledge of the amino acid residue networks involved in the allosteric communication will aid understanding GPCR signaling and the design of biased ligands. Angiotensin II type I receptor (AT1R) is an ideal model GPCR to study the molecular basis of ligand bias as it has multiple β-arrestin2 and Gq protein biased agonists as well as three-dimensional structures. Using Molecular Dynamics simulations, dynamic allostery analysis, and functional BRET assays, we identified a network of residues involved in allosteric communication from the angiotensin II binding site to the putative Gq coupling sites and another network to the β-arrestin2 coupling sites, with 6 residues common to both pathways located in TM3, TM5 and TM6. Our...
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Although multiple components of the cell membrane modulate the stability and activation of G protein coupled receptors (GPCRs), the activation mechanism comes from detergent studies, since it is challenging to study activation in... more
Although multiple components of the cell membrane modulate the stability and activation of G protein coupled receptors (GPCRs), the activation mechanism comes from detergent studies, since it is challenging to study activation in multi-component lipid bilayer. Using the multi-scale molecular dynamics simulations(50μs), our comparative study between cell membrane and detergents shows that: the changes in inter-residue distances, known as activation microswitches, show an ensemble of states in the extent of activation in cell membrane. We forward a rheostat model of GPCR activation rather than a binary switch model. Phosphatidylinositol bisphosphate (PIP2) and calcium ions, through a tug of war, maintain a balance between the GPCR stability and activity in cell membrane. Due to the lack of receptor stiffening effects by PIP2, detergents promote more transitions among conformational states than cell membrane. These findings connect the chemistry of cell membrane lipids to receptor acti...
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Research Interests: Pharmacology, Biochemistry, Biophysics, Chemistry, Inorganic Chemistry, and 13 moreMolecular Biology, Computational Biology, Virology, Cell Biology, Infectious Diseases, Medicine, Kinase, Binding Site, Biochemistry and cell biology, Allosteric regulation, Substrate Binding, ATP binding site, and Medical biochemistry and metabolomics
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Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the synovial joints. Although involvement of the fibroblast growth factor (FGF) signaling pathway has been suggested as an important modulator in RA... more
Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the synovial joints. Although involvement of the fibroblast growth factor (FGF) signaling pathway has been suggested as an important modulator in RA development, no clear evidence has been provided. In this study, we found that synovial fluid basic FGF (bFGF) concentration was significantly higher in RA than in osteoarthritis (OA) patients. bFGF stimulates proliferation and migration of human fibroblast-like synoviocytes (FLSs) by activation of the bFGF-FGF receptor 3 (FGFR3)-ribosomal S6 kinase 2 (RSK2) signaling axis. Moreover, a molecular docking study revealed that kaempferol inhibited FGFR3 activity by binding to the active pocket of the FGFR3 kinase domain. Kaempferol forms hydrogen bonds with the FGFR3 backbone oxygen of Glu555 and Ala558 and the side chain of Lys508. Notably, the inhibition of bFGF-FGFR3-RSK2 signaling by kaempferol suppresses the proliferation and migration of RA FLSs and the r...
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Research Interests: Biochemistry, Biophysics, Molecular Dynamics Simulation, Computational Biology, Kinetics, and 15 moreBiology, Biological Chemistry, Medicine, Protein Engineering, Biocatalysis, Fluorescence Resonance Energy Transfer, Biological Sciences, Humans, Mutation, Animals, Phosphorylation, Kinase, CHEMICAL SCIENCES, Protein Conformation, and Medical and Health Sciences
Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in... more
Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the bin...
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Stability of detergent-solubilized G-protein-coupled receptors (GPCRs) is crucial for their purification in a biologically relevant state, and it is well-known that short chain detergents such as octylglucoside are more denaturing than... more
Stability of detergent-solubilized G-protein-coupled receptors (GPCRs) is crucial for their purification in a biologically relevant state, and it is well-known that short chain detergents such as octylglucoside are more denaturing than long chain detergents such as dodecylmaltoside. However, the molecular basis for this phenomenon is poorly understood. To gain insights into the mechanism of detergent destabilization of GPCRs, we used atomistic molecular dynamics simulations of thermostabilized adenosine receptor (A2AR) mutants embedded in either a lipid bilayer or detergent micelles of alkylmaltosides and alkylglucosides. A2AR mutants in dodecylmaltoside or phospholipid showed low flexibility and good interhelical packing. In contrast, A2AR mutants in either octylglucoside or nonylglucoside showed decreased α-helicity in transmembrane regions, decreased α-helical packing, and the interpenetration of detergent molecules between transmembrane α-helices. This was not observed in octylg...
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G protein-coupled receptors (GPCRs) are highly dynamic and often denature when extracted in detergents. Deriving thermostable mutants has been a successful strategy to stabilize GPCRs in detergents, but this process is experimentally... more
G protein-coupled receptors (GPCRs) are highly dynamic and often denature when extracted in detergents. Deriving thermostable mutants has been a successful strategy to stabilize GPCRs in detergents, but this process is experimentally tedious. We have developed a computational method to predict the position of the thermostabilizing mutations for a given GPCR sequence. We have validated the method against experimentally measured thermostability data for single mutants of the β1-adrenergic receptor (β1AR), adenosine A2A receptor (A2AR) and neurotensin receptor 1 (NTSR1). To make these predictions we started from homology models of these receptors of varying accuracies and generated an ensemble of conformations by sampling the rigid body degrees of freedom of transmembrane helices. Then, an all-atom force field function was used to calculate the enthalpy gain, known as the "stability score" upon mutation of every residue, in these receptor structures, to alanine. For all three...
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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... more
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 -arrestin-biased agonists...
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The neurotensin receptor NTSR1 binds the peptide agonist neurotensin (NTS) and signals preferentially via the Gq protein. Recently Grisshammer and coworkers reported the crystal structure of a thermostable mutant NTSR1-GW5 with NTS bound.... more
The neurotensin receptor NTSR1 binds the peptide agonist neurotensin (NTS) and signals preferentially via the Gq protein. Recently Grisshammer and coworkers reported the crystal structure of a thermostable mutant NTSR1-GW5 with NTS bound. Understanding how the mutations thermostabilize the structure would allow efficient design of thermostable mutant GPCRs for protein purification, and subsequent biophysical studies. Using microsecond scale molecular dynamics simulations (4 μs) of the thermostable mutant NTSR1-GW5 and wild type NTSR1, we have elucidated the structural and energetic factors that affect the thermostability and dynamics of NTSR1. The thermostable mutant NTSR1-GW5 is found to be less flexible and less dynamic than the wild type NTSR1. The point mutations confer thermostability by improving the inter-helical hydrogen bonds, hydrophobic packing, and the receptor interactions with the lipid bilayer, especially in the intracellular regions. During MD, NTSR1-GW5 becomes more...