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 dimerization affinity with mutational studies, in which we identify key residues responsible for dimerization. To connect the dimerization affinity to its functional consequences we also measured the spectral shifts in the absorption profiles of the wild-type and mutant cone opsins regenerated with the retinal chromophore. The results of our work provide critical insight into the molecular basis of color vision. They also will inform the ongoing study of GPCR dimerization in cell signaling. 2925-Pos Board B302 Ligand-Induced Growth of CD36-Fyn Clusters Induces Signaling John Maringa Githaka1, Anthony R. Vega2, Michelle A. Baird3, Michael W. Davidson3, Khuloud Jaqaman2, Nicolas Touret1. 1 Biochemistry, University of Alberta, Edmonton, AB, Canada, 2Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA, 3 National High Magnetic Field Laboratory and Biological Science, Florida State University, Tallahassee, FL, USA. Nanoclustering is emerging as a key organizational principle of membrane proteins. Using superresolution imaging, we investigated the molecular organization of the clustering-responsive receptor CD36 and its downstream effector Fyn in response to thrombospondin-1 (TSP-1), an anti-angiogenic ligand that promotes endothelial cells apoptosis. At steady state, CD36 receptors preexist in nanoclusters (diameter of about 100 nm). Fyn is already present in these clusters, and its N-terminal membrane targeting domain is sufficient for this association. Even if already associated, we found that Fyn only becomes activated activated upon TSP-1 binding through an enhancement of CD36-Fyn clustering, forming larger and denser clusters. These enhancements are supported by the actin cytoskeleton and the presence of plasma membrane cholesterol as their perturbation abolishes signaling. Our data demonstrate cooperation between cholesterol-dependent domains and the cortical actin cytoskeleton in the reorganization of the receptor-effector pair CD36-Fyn that enables signaling during TSP-1 stimulation. 2926-Pos Board B303 Distinct Roles of a-Actinin in Regulating Talin-Induced Activation of aIIbb3 versus a5b1 Integrins Hengameh Shams, Mohammad R.K. Mofrad. UC Berkeley, Berkeley, CA, USA. Cell adhesion and signaling is crucial for various cellular functions including differentiation, proliferation and growth. Integrins are key membrane receptors that are central to the formation of adhesion sites and their function is regulated through binding with talin. However, interactions of integrin and talin with other molecules can modify and in some cases inhibit the course of integrin activation. a-Actinin is a unique protein that both crosslinks actin filaments and plays an important role in adhesion formation and maturation. A previous study showed that a-actinin has distinct roles in regulating talininduced activation of aIIbb3 and a5b1 integrins, i.e. it prevents activation for aIIbb3 and promotes adhesion for a5b1. We generated all-atomic models of a-actinin, talin and both types of integrin dimers, aIIbb3 and a5b1, in order to study the underlying mechanisms of their interplay. Our results revealed an interesting competition between talin and a-actinin for the same residues on the aIIbb3 integrin tail, which was in agreement with the previously reported binding site. Also, our models showed simultaneous interactions of a-actinin and talin with integrin a5b1 and an exciting cooperative interaction between them. Consequently, our results shed light on fundamental mechanisms of force transmission that are critical for integrin-mediated insideout signaling. 2927-Pos Board B304 Targeting Liposomes for Uptake into CEACAM-Expressing Human Cells using a Bacterial Membrane Protein Jason Kuhn1, Asya Smirnov2, Alison K. Criss2, Linda Columbus1. 1 Department of Chemistry, University of Virginia, Charlottesville, VA, USA, 2 Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA. The pathogenic bacteria Neisseria gonorrhoeae and N. meningitidis induce their own phagocytosis into human host cells prior to replication. Bacterial cell entry is promoted by the binding of Neisserial outer membrane opacityassociated (Opa) proteins to human carcinoembryonic antigen-like cell adhesion molecule (CEACAM) receptors, a widely-distributed class of cell receptors in the human body. Binding of Opa proteins to either CEACAM1, CEACAM3, CEACAM5, or CEACAM6 generates intracellular signalling events which lead to bacterial internalization by both phagocytes and epithelial cells. We are interested in determining whether purified and folded Opa proteins stabilized in liposomes retain the ability to promote liposomal entry into epithelial cells similar to Neisseria. The ability to target CEACAMþ cells
for Opa proteoliposome uptake may prove valuable in targeted therapeutic delivery. Our results demonstrate that Opa-proteoliposomes target surface CEACAM receptors on transfected HeLa cells for binding and preliminary data indicate the liposomes are taken into the cells. This internalization is dependent on metabolic energy, suggesting the cells may employ active-uptake mechanisms to internalize the liposomes. Our work shows that Opa proteoliposomes deserve further consideration as a platform for targeted delivery to CEACAMexpressing cells. 2928-Pos Board B305 Mechanism of Interaction between Adenosine Phosphates and Lysenin Channels Sheenah L. Bryant1, Nisha Shrestha1, Paul Carnig2, Samuel R. Kosydar2, Philip Belzeski2, Jason May2, Lauren McDaid3, Daniel Fologea4. 1 Biomolecular Science, Boise State University, Boise, ID, USA, 2Physics, Boise State University, Boise, ID, USA, 3Biology, Boise State University, Boise, ID, USA, 4Biomolecular Science PhD Program/Department of Physics, Boise State University, Boise, ID, USA. Lysenin, a 297 amino acid pore forming toxin extracted from the earthworm E. foetida, inserts hexameric channels in natural and artificial membranes containing sphingomyelin. A distinct feature of lysenin channels is their asymmetric voltage induced gating which occurs at low positive transmembrane potentials. Although the structure of the channel is not yet solved, previous studies of the lysenin monomeric structure and interaction with membranes indicate the presence of local positive charges within the protein’s structure. Consequently, we hypothesized that highly charged organic anions such as ATP, ADP, and AMP may interact with the channel and alter its functionality. Here we show that the macroscopic conductance of the channels was diminished by addition of various adenosine phosphates in a charge and concentration-dependent manner. Single channel explorations have shown a gradual reduction of the ionic currents indicating interaction with absence of gating. Buffer-exchange experiments demonstrated that the current inhibition is reversible, pointing out a potential electrostatic mechanism of interaction between channels and anions which implies binding. This hypothesis was further sustained by analyzing the inhibitory effects of ATP comprising ionic solutions with different ionic strengths. Hill analysis provided further insights into the inhibition process and indicated a positive cooperativity between binding events. In addition, the I-V plots recorded in the presence of increasing inhibitor concentration demonstrate a significant rightward shift of the voltage-induced gating profile. The reduced effects elicited by AMP as compared with ATP and ADP, the reversibility of the inhibition, and the strong dependency of the observed effects on the ionic strength point out an electrostatic mechanism of interaction which may yield partial occlusion of the channels and modulation of the voltage-induced gating. This work sets an example of purinergic signaling that implies modulating the functionality of an exogenous membrane transporter.
Exocytosis and Endocytosis II 2929-Pos Board B306 Imaging the Rapid Recruitment of Dynamins at the Exocytic Fusion Pore Adam J. Trexler, Justin W. Taraska. National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA. The size and shape of the exocytic fusion pore directly modulates the release of vesicle cargoes like insulin, hormones, and neurotransmitters. Though a number of proteins have been implicated in fusion pore regulation, the molecular details of this process and how these proteins work together to shape the membrane are largely unknown. Using TIRF microscopy in living INS-1 cells we directly image the coincident and transient recruitment of dynamin proteins, the regulatory lipid PIP2, and BAR domain proteins amphiphysin, syndapin, and endophilin to exocytic sites. Recruitment occurs at the moment of membrane fusion. Evidence from imaging and siRNA depletion of endogenous dynamins suggests that dynamin-1 and dynamin-2 have distinct functional roles at sites of exocytosis. We show that perturbing dynamin-1 interaction with PIP2 or amphiphysin blocks its recruitment to exocytic sites and has functional effects on vesicle cargo release. We turn to stimulated emission depletion (STED) super-resolution fluorescence microscopy to directly visualize the organization of dynamins at sites of exocytosis in living cells. The spatiotemporal coincident recruitment of dynamins, PIP2, and BAR domain proteins, and their known interactions, strongly suggest these molecules form a complex at the nascent exocytic fusion pore. Such a protein and lipid assembly represents a novel paradigm for controlling fusion pore size and shape and directly modulating endocrine cargo release.