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Mechanism of TIM1, TIM3, and TIM4 Binding to Lipid Membranes
592a
Wednesday, March 2, 2016
signaling pathway from genetic feedback loops can be modified by receptors clustering. Our method demonstrates the functional importance of spatial organization in cross-membrane signal transduction. 2920-Pos Board B297 Tracking Changes in Protonation and Conformation during Photoactivation of a Phytochrome Protein Serena Donnini, Modi Vaibhav, Janne Ihalainen, Gerrit Groenhof. The Nanoscience Center and the Department of Biological and Environmental Science, University of Jyvaskyla, Jyvaskyla, Finland. Phytochromes are photosensor proteins in plants and bacteria. The biological response is mediated by structural changes that follow photon absorption in the protein complex. The initial step is the photoisomerization of the biliverdin chromophore. How this leads to large-scale structural changes of the whole complex is, however, poorly understood. In this work, we use molecular dynamics (MD) simulations to investigate the structural changes after isomerization. In particular, we perform MD simulations at constant pH, using a recently developed method, to explore the effect of chromophore isomerization on the protonation (pKa) of nearby residues. In addition, we use a hybrid quantum mechanics/molecular mechanics approach to investigate the effect of isomerization, protonation and protein conformational changes on the absorption spectrum of the protein, for which experimental data are available. Here, we will first describe the constant pH MD simulations, and then compare the calculated spectra to experiment, and discuss the implications of our results for the photo-switching mechanism. 2921-Pos Board B298 Mechanism of TIM1, TIM3, and TIM4 Binding to Lipid Membranes Zhiliang Gong1, Daniel Kerr2, Gregory T. Tietjen3, James Michael Henderson1, Adrienne M. Luoma4, Wei Bu5, Kathleen D. Cao1, Hyeondo Luke Hwang1, Theodore L. Steck6, Binhua Lin5, Erin J. Adams4, Ka Yee C. Lee1. 1 Department of Chemistry, University of Chicago, Chicago, IL, USA, 2 Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 3School of Engineering & Applied Science, Yale University, Chicago, IL, USA, 4Committee on Immunology and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA, 5Center for Advanced Radiation Sources, University of Chicago, Chicago, IL, USA, 6 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA. T cell/transmembrane immunoglobulin mucin protein 1, 3, and 4 (TIM1, TIM3, and TIM4) are vital regulators in the innate immune system. Their activation involves specifically binding phosphatidylserine (PS) exposed cell membranes. Elucidation of the detailed mechanism of this TIMs/lipid interaction is key to understanding their immunological functions. Here we have established the binding force between the TIMs and lipid membrane to be composed of three components, namely the protein/Ca2þ/PS coordination chemistry, hydrophobic insertion, and electrostatic attraction. We have further quantified these three forces in terms of association/dissociation constants in different large unilamellar vesicle model systems, using fluorescence spectral shift as the tool. Our quantitative analysis has led to the surprising discovery of a three- to fourfold affinity enhancement effect of minute amounts of the negatively charged phosphatidic acid (PA) are present in the membrane for TIM3, while much attenuated effects were observed for TIM1 and TIM4, suggesting the level of PA might play an important role in TIM3 regulation. TIM4 binds to PS containing membranes most potently, yielding a TIM4/PS stoichiometry of around 4.5/1, which agrees well with our model that 4 peripheral basic amino acid residues around the PS coordination pocket contribute to binding via binding to negative lipid headgroups on the membrane. We have studied the concentration of Ca2þ at the interface between a lipid monolayer and the subphase on a Langmuir trough using x-ray fluorescence to find significant accumulation, suggesting a cloud of Ca2þ might present an attractive force pulling the TIMs toward the membrane. 2922-Pos Board B299 Non Bacterial Lipid and Proteins Aggregates are Activators of the Innate System Jean-Marie Ruysschaert, Malvina Pizzuto, Caroline Lonez. Structure and functions of biological membranes, Free University of Brussels, Brussels, Belgium. Toll like receptors (TLRs) are classically recognised by bacterial and viral components that induce the secretion of inflammatory cytokines. It was recently demonstrated however that non bacterial molecules like synthetic nanoparticules (gold, carbon nanoparticules) and amyloid structures do activate the innate system through the same TLRs. We will illustrate this new concept
with a few examples and discussed of the consequences of such an unexpected activation. 1) Lipid nanoparticles activate TLRs via a new binding site different from the one identified so far for natural ligands. 2) TLRs are activated by amyloid structures and this activation is structure dependent. The induction of inflammatory responses by these fibrillar aggregates is linked to their intrinsic structure (not to a sequence).It is therefore tempting to speculate that amyloid fibrils represent a new class of danger signals detected by the innate machinery through sensing of their common cross-b structure that does not exist in any other proteins so far. Our observations suggest that the cross-b structural signature of amyloid fibrils is a generic signal that activate immunity for all neurodegenerative diseases (Alzheimer, Parkinson).Interestingly oligomers which are considered has highly cytotoxic do not activate. It has been suggested that a therapeutic that blocks the activity of the inflammatory process might effectively interfere with the progression of Alzheimer disease. These inflammatory reactions can be desired (for vaccine development), unwanted (for delivery applications) or involved in the induction of noninfectious diseases (amyloidoses, prion-related diseases). For that reason, development of new molecules targeting or inhibiting these inflammatory responses may lead to therapeutic perspectives largely unintended until now. 2923-Pos Board B300 A Cytokine Receptor Revolution: Activation of the Type-I Cytokine Receptors via Protomer Rotation Michael Corbett1, David Poger1, Alan E. Mark1,2. 1 SCMB, The University of Queensland, Brisbane, Australia, 2Institute for Molecular Bioscience, Brisbane, Australia. The Type-I Cytokine Receptors regulate diverse cellular functions that encompass immune responses, growth, metabolism, lactation and red blood cell production. A sub-group of receptors that are models for receptor activation includes the growth hormone receptor (GHR), prolactin receptor (PRLR) and erythropoietin receptor (EPOR). While the cellular effects of receptor activation have been well characterised, the atomic details of how these receptors mechanically couple cytokine binding to the extracellular domains through the plasma membrane has remained elusive. Using fully atomistic molecular dynamics simulations, we have shown the extracellular domains of the GHR and PRLR homodimers undergo a relative rigid body rotation upon activation. Recently, we have tested if the rotation-based mechanism could be extended to erythropoietin (EPO) induced activation of the EPOR homodimer along with agonistic and antagonistic EPO mimetic peptides. In the simulations, the EPOR protomers rotate as rigid bodies similarly to the GHR and PRLR upon cytokine activation. However, analysis of the mimetic-bound EPOR could not distinguish between agonised or antagonised conformations of the dimer. Simulations of the GHR, PRLR and EPOR embedded in membranes exhibited different behavior in cholesterol rich membranes, suggesting a key role of cholesterol in receptor activation. The simulations demonstrate a shared cytokine induced rotational-based mechanism of activation of Type-I Cytokine Receptors. The atomic resolution of the mechanical coupling of the receptors through the plasma membrane provides clearer understanding of how the signal of cytokine binding is transmitted from the extracellular to the cytosolic domains. 2924-Pos Board B301 Probing the Dimerization Affinity of Visual Opsins Megan J. Kaliszewski1, William D. Comar1, Kevin C. Skinner1, Beata Jastrzebska2, Krzysztof Palczewski2, Adam W. Smith1. 1 Chemistry, University of Akron, Akron, OH, USA, 2Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA. Visual opsins are the light sensing proteins at the center of human vision. Rhodopsin is the pigment for low-light vision, and three cone opsins serve as the pigments for daytime image-forming vision. Opsin proteins are part of the G protein-coupled receptor (GPCR) family, and rhodopsin has served as something like a prototype for structural studies of GPCRs. Rhodopsin has also been observed to form dimeric complexes in vitro, in tissue, and in cell cultures, including recent work from our lab using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Here we report on our recent investigation of cone opsin dimerization in a heterologous expression system. PIE-FCCS is used to resolve the relative population of dimeric complexes over a range of concentrations in live cell membranes. Surprisingly, the homodimerization affinities of the cone opsins are not identical to rhodopsin and there are significant differences in the dimerization affinity of the three cone opsins. This raises important questions about the physiological role of opsin dimerization, which has wide-ranging implications for GPCR dimerization in general. To investigate these questions we explored the differential
Wednesday, March 2, 2016
signaling pathway from genetic feedback loops can be modified by receptors clustering. Our method demonstrates the functional importance of spatial organization in cross-membrane signal transduction. 2920-Pos Board B297 Tracking Changes in Protonation and Conformation during Photoactivation of a Phytochrome Protein Serena Donnini, Modi Vaibhav, Janne Ihalainen, Gerrit Groenhof. The Nanoscience Center and the Department of Biological and Environmental Science, University of Jyvaskyla, Jyvaskyla, Finland. Phytochromes are photosensor proteins in plants and bacteria. The biological response is mediated by structural changes that follow photon absorption in the protein complex. The initial step is the photoisomerization of the biliverdin chromophore. How this leads to large-scale structural changes of the whole complex is, however, poorly understood. In this work, we use molecular dynamics (MD) simulations to investigate the structural changes after isomerization. In particular, we perform MD simulations at constant pH, using a recently developed method, to explore the effect of chromophore isomerization on the protonation (pKa) of nearby residues. In addition, we use a hybrid quantum mechanics/molecular mechanics approach to investigate the effect of isomerization, protonation and protein conformational changes on the absorption spectrum of the protein, for which experimental data are available. Here, we will first describe the constant pH MD simulations, and then compare the calculated spectra to experiment, and discuss the implications of our results for the photo-switching mechanism. 2921-Pos Board B298 Mechanism of TIM1, TIM3, and TIM4 Binding to Lipid Membranes Zhiliang Gong1, Daniel Kerr2, Gregory T. Tietjen3, James Michael Henderson1, Adrienne M. Luoma4, Wei Bu5, Kathleen D. Cao1, Hyeondo Luke Hwang1, Theodore L. Steck6, Binhua Lin5, Erin J. Adams4, Ka Yee C. Lee1. 1 Department of Chemistry, University of Chicago, Chicago, IL, USA, 2 Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 3School of Engineering & Applied Science, Yale University, Chicago, IL, USA, 4Committee on Immunology and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA, 5Center for Advanced Radiation Sources, University of Chicago, Chicago, IL, USA, 6 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA. T cell/transmembrane immunoglobulin mucin protein 1, 3, and 4 (TIM1, TIM3, and TIM4) are vital regulators in the innate immune system. Their activation involves specifically binding phosphatidylserine (PS) exposed cell membranes. Elucidation of the detailed mechanism of this TIMs/lipid interaction is key to understanding their immunological functions. Here we have established the binding force between the TIMs and lipid membrane to be composed of three components, namely the protein/Ca2þ/PS coordination chemistry, hydrophobic insertion, and electrostatic attraction. We have further quantified these three forces in terms of association/dissociation constants in different large unilamellar vesicle model systems, using fluorescence spectral shift as the tool. Our quantitative analysis has led to the surprising discovery of a three- to fourfold affinity enhancement effect of minute amounts of the negatively charged phosphatidic acid (PA) are present in the membrane for TIM3, while much attenuated effects were observed for TIM1 and TIM4, suggesting the level of PA might play an important role in TIM3 regulation. TIM4 binds to PS containing membranes most potently, yielding a TIM4/PS stoichiometry of around 4.5/1, which agrees well with our model that 4 peripheral basic amino acid residues around the PS coordination pocket contribute to binding via binding to negative lipid headgroups on the membrane. We have studied the concentration of Ca2þ at the interface between a lipid monolayer and the subphase on a Langmuir trough using x-ray fluorescence to find significant accumulation, suggesting a cloud of Ca2þ might present an attractive force pulling the TIMs toward the membrane. 2922-Pos Board B299 Non Bacterial Lipid and Proteins Aggregates are Activators of the Innate System Jean-Marie Ruysschaert, Malvina Pizzuto, Caroline Lonez. Structure and functions of biological membranes, Free University of Brussels, Brussels, Belgium. Toll like receptors (TLRs) are classically recognised by bacterial and viral components that induce the secretion of inflammatory cytokines. It was recently demonstrated however that non bacterial molecules like synthetic nanoparticules (gold, carbon nanoparticules) and amyloid structures do activate the innate system through the same TLRs. We will illustrate this new concept
with a few examples and discussed of the consequences of such an unexpected activation. 1) Lipid nanoparticles activate TLRs via a new binding site different from the one identified so far for natural ligands. 2) TLRs are activated by amyloid structures and this activation is structure dependent. The induction of inflammatory responses by these fibrillar aggregates is linked to their intrinsic structure (not to a sequence).It is therefore tempting to speculate that amyloid fibrils represent a new class of danger signals detected by the innate machinery through sensing of their common cross-b structure that does not exist in any other proteins so far. Our observations suggest that the cross-b structural signature of amyloid fibrils is a generic signal that activate immunity for all neurodegenerative diseases (Alzheimer, Parkinson).Interestingly oligomers which are considered has highly cytotoxic do not activate. It has been suggested that a therapeutic that blocks the activity of the inflammatory process might effectively interfere with the progression of Alzheimer disease. These inflammatory reactions can be desired (for vaccine development), unwanted (for delivery applications) or involved in the induction of noninfectious diseases (amyloidoses, prion-related diseases). For that reason, development of new molecules targeting or inhibiting these inflammatory responses may lead to therapeutic perspectives largely unintended until now. 2923-Pos Board B300 A Cytokine Receptor Revolution: Activation of the Type-I Cytokine Receptors via Protomer Rotation Michael Corbett1, David Poger1, Alan E. Mark1,2. 1 SCMB, The University of Queensland, Brisbane, Australia, 2Institute for Molecular Bioscience, Brisbane, Australia. The Type-I Cytokine Receptors regulate diverse cellular functions that encompass immune responses, growth, metabolism, lactation and red blood cell production. A sub-group of receptors that are models for receptor activation includes the growth hormone receptor (GHR), prolactin receptor (PRLR) and erythropoietin receptor (EPOR). While the cellular effects of receptor activation have been well characterised, the atomic details of how these receptors mechanically couple cytokine binding to the extracellular domains through the plasma membrane has remained elusive. Using fully atomistic molecular dynamics simulations, we have shown the extracellular domains of the GHR and PRLR homodimers undergo a relative rigid body rotation upon activation. Recently, we have tested if the rotation-based mechanism could be extended to erythropoietin (EPO) induced activation of the EPOR homodimer along with agonistic and antagonistic EPO mimetic peptides. In the simulations, the EPOR protomers rotate as rigid bodies similarly to the GHR and PRLR upon cytokine activation. However, analysis of the mimetic-bound EPOR could not distinguish between agonised or antagonised conformations of the dimer. Simulations of the GHR, PRLR and EPOR embedded in membranes exhibited different behavior in cholesterol rich membranes, suggesting a key role of cholesterol in receptor activation. The simulations demonstrate a shared cytokine induced rotational-based mechanism of activation of Type-I Cytokine Receptors. The atomic resolution of the mechanical coupling of the receptors through the plasma membrane provides clearer understanding of how the signal of cytokine binding is transmitted from the extracellular to the cytosolic domains. 2924-Pos Board B301 Probing the Dimerization Affinity of Visual Opsins Megan J. Kaliszewski1, William D. Comar1, Kevin C. Skinner1, Beata Jastrzebska2, Krzysztof Palczewski2, Adam W. Smith1. 1 Chemistry, University of Akron, Akron, OH, USA, 2Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA. Visual opsins are the light sensing proteins at the center of human vision. Rhodopsin is the pigment for low-light vision, and three cone opsins serve as the pigments for daytime image-forming vision. Opsin proteins are part of the G protein-coupled receptor (GPCR) family, and rhodopsin has served as something like a prototype for structural studies of GPCRs. Rhodopsin has also been observed to form dimeric complexes in vitro, in tissue, and in cell cultures, including recent work from our lab using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Here we report on our recent investigation of cone opsin dimerization in a heterologous expression system. PIE-FCCS is used to resolve the relative population of dimeric complexes over a range of concentrations in live cell membranes. Surprisingly, the homodimerization affinities of the cone opsins are not identical to rhodopsin and there are significant differences in the dimerization affinity of the three cone opsins. This raises important questions about the physiological role of opsin dimerization, which has wide-ranging implications for GPCR dimerization in general. To investigate these questions we explored the differential