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Investigating MUC1 Transmembrane Dimer Structure using Replica Exchange Molecular Dynamics
Sunday, February 12, 2017 G protein-coupled receptors (GPCRs) and G proteins are key players of cellular signal transduction. They transduce signals from a multitude of extracellular stimuli into the cells. Despite a number of studies, many aspects of molecular mechanisms and spatiotemporal dynamics of G protein interactions with GPCRs remain unclear. In particular, it is uncertain whether G proteins can form complexes with inactive GPCRs, and what is the stability and functional significance of such complexes. In order to address these issues, we have investigated the interactions between the G proteins and various GPCRs using the technique of twophoton polarization microscopy (2PPM). 2PPM, developed in our laboratory, allows sensitive monitoring of protein conformational changes and protein-protein interactions, in living cells, in real time, using a single fluorescent protein tag. Our results demonstrate that 2PPM indeed allows observing interactions between G proteins and representative GPCRs, using a single fluorescent protein tag. Our experiments reveal an important role of receptor basal activity for GPCR - G protein interactions, yielding insights into receptor - G protein precoupling and other aspects of GPCR - G protein signal transduction. 453-Pos Board B218 Investigating MUC1 Transmembrane Dimer Structure using Replica Exchange Molecular Dynamics Christina M. Freeman, Alexander J. Sodt. NICHD, National Institutes of Health, Bethesda, MD, USA. The epithelial single-pass transmembrane glycoprotein Mucin 1 (MUC1) is a main constituent of mucus and has roles in cell signaling and differentiation. In addition, overexpression and subsequent homodimerization of the C-terminal subunit of MUC1 has been implicated in the progression of many cancers, particularly breast cancer. Recent experiments have shown that strong dimerization is dependent on the formation of disulfide bonds between two cysteine residues in the juxtamembrane region, but weak dimers can also be formed without them. Certain mutations in the TMD can also partially disrupt the strength of the dimer. This suggests that protein-protein or protein-lipid interactions are also mediating dimer formation. However, the transmembrane domain of MUC1-C doesn’t appear to contain common oligomerization motifs such as the Sm-X3-Sm sequence found in glycophorin A and many receptor tyrosine kinases. Understanding the physical mechanisms by which MUC1-C dimerizes within the surrounding plasma membrane environment will help in creating drug targets, as well as generating a more comprehensive model of protein-lipid interactions. To investigate this, atomistic replica exchange molecular dynamics simulations are being used to find stable conformations of wild-type and mutant MUC1 TMDs. 454-Pos Board B219 Electrostatics Facilitates the Trimer-of-Dimers Formation of the Chemoreceptor Signaling Domain Marharyta Petukh1, Davi Ortega2, Igor B. Zhulin1. 1 UTK/ORNL, Oak Ridge, TN, USA, 2The California Institute of Technology, Pasadena, CA, USA. Chemoreceptors are crucial components of the bacterial sensory system that modulates cellular motility. They detect changes in the environment and transmit information to CheA histidine kinase, which ultimately controls cellular flagellar motors. The prototypical Tsr chemoreceptor in E. coli is a homodimer containing two principle functional modules: (i) a periplasmic ligand-binding domain and (ii) a cytoplasmic signaling domain comprising an antiparallel, four-helix coiled-coil bundle. Receptor dimers are arranged into a trimer-of-dimers, which is a minimal physical unit essential for enhancing the CheA activity several hundredfold. Recent advances in cryo-electron tomography showed that trimers-of-dimers are arranged into highly ordered hexagon arrays at the cell pole; however, the mechanism underlying the trimer-of-dimer and higher order array formation remains unknown. Current evidence from structural and biochemical studies suggest that trimers-of-dimers are maintained exclusively by contacts at the chemoreceptor cytoplasmic tip. Here, using all-atom, microsecond-range MD simulation of the Tsr trimer-of-dimers crystal structure, we show that dimers within the trimer may interact throughout the entire length of the signaling domain. While inter-dimer contacts at the chemoreceptor tip occur via hydrophobic interactions, complete dimer ‘‘zipping’’ is facilitated by electrostatics, especially by the polar solvation component. We also show that many of the residues involved in establishing hydrogen bonds and salt bridges between dimers are evolutionary conserved.
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455-Pos Board B220 Investigating Initial Events of IgE Receptor Signaling with SuperResolution Microscopy and Monte Carlo Simulations Eshan Mitra1, James P. Sethna2, David Holowka1, Barbara Baird1. 1 Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA, 2 Physics, Cornell University, Ithaca, NY, USA. The high affinity IgE receptor (FcεRI) in mast cells plays a central role in initiating allergic responses, and also serves as a model system for immune receptor signaling. FcεRI binds IgE antibody, which confers specificity for antigen. The cross-linking of IgE-FcεRI by antigen stimulates a transmembrane signal that leads to downstream events including Ca2þ mobilization and degranulation. Our work seeks to understand the molecular basis of these initial signaling events. To examine signal initiation experimentally, we performed PALM/STORM super-resolution imaging. We employed structurally defined ligands to gain molecular-level control over the structure of the IgE cluster formed. Two trivalent ligands studied, dsDNA based Y16 and Y46, differ in the spacing of receptor binding sites. With one-color STORM on live cells, we imaged the dynamic clustering of receptors upon stimulation. We found that Y16 and Y46 differ in the density of receptors in clusters formed, as quantified by autocorrelation functions. With two-color PALM/STORM, we quantified the extent to which the differing structures of these IgE clusters affect their capacity to recruit other membrane components, including signaling partner Lyn kinase and markers for liquid ordered (Lo) membrane phase. We used theoretical modeling to further address the potential role of membrane lipids in promoting the recruitment of a kinase to a cluster of receptors. We modeled the membrane with a 2D Ising model, and calculated the change in free energy associated with recruiting a Lo-preferring kinase into a Lopreferring receptor cluster. Using this framework, we asked what structural features of a receptor cluster are most important for effective lipid-mediated signaling. Ongoing work uses an extension of the Ising model in order to address other models of membrane phase behavior, including microemulsions. 456-Pos Board B221 Fluorescence Fluctuation Spectroscopy of Dopaminergic Signaling in Pancreatic Beta Cells Daniel J.P. Foust, Alessandro Ustione, David W. Piston. Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA. Dopamine inhibits insulin secretion from pancreatic beta cells via D2-like dopamine receptors and we seek to understand the molecular details of this inhibition using the tools of fluorescence fluctuation spectroscopy. Normal insulin secretion is critical to maintaining blood glucose homeostasis and the prevention of type II diabetes. Although glucose stimulated insulin secretion is well understood, there are hundreds of receptors expressed in beta cells, including G-protein coupled dopamine receptors, which may provide additional mechanisms for regulating insulin secretion. Previously, we have shown that D2-like (D2, D3, D4) dopamine receptors confer beta cell sensitivity to dopamine. Furthermore, we demonstrated that the D3 homolog is the receptor primarily responsible for dopamine sensitivity, although the other receptors may compensate in a D3-knockout scenario. Electrophysiology has revealed an inwardly-rectified current in dopamine-stimulated beta cells, implicating ion channels as possible downstream signaling targets. G-protein coupled inwardly rectifying Kþ (GIRK) channels are known effectors of D2-like receptors, although transactivation of GIRK channels by dopamine receptors has not been demonstrated explicitly in beta cells. GIRK channels are activated by tetrameric binding of Gbg subunits dissociated from the trimeric G-protein complex. To study dopamine receptor interactions with downstream effectors we express fluorescently labeled proteins (receptors, G-proteins, channels) in MIN6 cells. Cells are imaged with single- or two-photon excitation and these images are analyzed by considering the intensity distribution (photon counting histogram) and correlation (image correlation spectroscopy). Using these analyses we show that dopamine receptors co-cluster with G-proteins at discrete sites on the plasma membrane. At these cluster sites, the apparent brightness of labeled Gbg subunits increases with dopamine stimulus. Brightness increases indicate oligomerization, therefore these results support our hypothesis that dopamine receptor activation promotes the opening of GIRK channels via tetrameric binding of dissociated Gbg subunits to channels. 457-Pos Board B222 Dynamics of Various Phospholipase C-B Complexes Ashima Singla, Suzanne Scarlata. Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, MA, USA.
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455-Pos Board B220 Investigating Initial Events of IgE Receptor Signaling with SuperResolution Microscopy and Monte Carlo Simulations Eshan Mitra1, James P. Sethna2, David Holowka1, Barbara Baird1. 1 Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA, 2 Physics, Cornell University, Ithaca, NY, USA. The high affinity IgE receptor (FcεRI) in mast cells plays a central role in initiating allergic responses, and also serves as a model system for immune receptor signaling. FcεRI binds IgE antibody, which confers specificity for antigen. The cross-linking of IgE-FcεRI by antigen stimulates a transmembrane signal that leads to downstream events including Ca2þ mobilization and degranulation. Our work seeks to understand the molecular basis of these initial signaling events. To examine signal initiation experimentally, we performed PALM/STORM super-resolution imaging. We employed structurally defined ligands to gain molecular-level control over the structure of the IgE cluster formed. Two trivalent ligands studied, dsDNA based Y16 and Y46, differ in the spacing of receptor binding sites. With one-color STORM on live cells, we imaged the dynamic clustering of receptors upon stimulation. We found that Y16 and Y46 differ in the density of receptors in clusters formed, as quantified by autocorrelation functions. With two-color PALM/STORM, we quantified the extent to which the differing structures of these IgE clusters affect their capacity to recruit other membrane components, including signaling partner Lyn kinase and markers for liquid ordered (Lo) membrane phase. We used theoretical modeling to further address the potential role of membrane lipids in promoting the recruitment of a kinase to a cluster of receptors. We modeled the membrane with a 2D Ising model, and calculated the change in free energy associated with recruiting a Lo-preferring kinase into a Lopreferring receptor cluster. Using this framework, we asked what structural features of a receptor cluster are most important for effective lipid-mediated signaling. Ongoing work uses an extension of the Ising model in order to address other models of membrane phase behavior, including microemulsions. 456-Pos Board B221 Fluorescence Fluctuation Spectroscopy of Dopaminergic Signaling in Pancreatic Beta Cells Daniel J.P. Foust, Alessandro Ustione, David W. Piston. Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA. Dopamine inhibits insulin secretion from pancreatic beta cells via D2-like dopamine receptors and we seek to understand the molecular details of this inhibition using the tools of fluorescence fluctuation spectroscopy. Normal insulin secretion is critical to maintaining blood glucose homeostasis and the prevention of type II diabetes. Although glucose stimulated insulin secretion is well understood, there are hundreds of receptors expressed in beta cells, including G-protein coupled dopamine receptors, which may provide additional mechanisms for regulating insulin secretion. Previously, we have shown that D2-like (D2, D3, D4) dopamine receptors confer beta cell sensitivity to dopamine. Furthermore, we demonstrated that the D3 homolog is the receptor primarily responsible for dopamine sensitivity, although the other receptors may compensate in a D3-knockout scenario. Electrophysiology has revealed an inwardly-rectified current in dopamine-stimulated beta cells, implicating ion channels as possible downstream signaling targets. G-protein coupled inwardly rectifying Kþ (GIRK) channels are known effectors of D2-like receptors, although transactivation of GIRK channels by dopamine receptors has not been demonstrated explicitly in beta cells. GIRK channels are activated by tetrameric binding of Gbg subunits dissociated from the trimeric G-protein complex. To study dopamine receptor interactions with downstream effectors we express fluorescently labeled proteins (receptors, G-proteins, channels) in MIN6 cells. Cells are imaged with single- or two-photon excitation and these images are analyzed by considering the intensity distribution (photon counting histogram) and correlation (image correlation spectroscopy). Using these analyses we show that dopamine receptors co-cluster with G-proteins at discrete sites on the plasma membrane. At these cluster sites, the apparent brightness of labeled Gbg subunits increases with dopamine stimulus. Brightness increases indicate oligomerization, therefore these results support our hypothesis that dopamine receptor activation promotes the opening of GIRK channels via tetrameric binding of dissociated Gbg subunits to channels. 457-Pos Board B222 Dynamics of Various Phospholipase C-B Complexes Ashima Singla, Suzanne Scarlata. Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, MA, USA.