Introducing Improved Protein Side Chain Dynamics in the MARTINI Model to Simulate Protein-Membrane Interactions

Wednesday, March 2, 2016 The bulk of hsp function occurred within the cytosol and subcellular compartments. However, some hsp have also been found outside cells released by an active mechanism independent of cell death. Extracellular hsp act as signaling molecules directed at activating a systemic response to stress. The export of hsp requires the translocation from the cytosol into the extracellular milieu across the plasma membrane. We have proposed that membrane insertion is the initial step in the export process. We investigated the interaction of the major inducible hsp, Hsp70, from humans (HspA1) and bacteria (DnaK) with liposomes. We found that HspA1 displayed a high specificity for negatively charged phospholipids, such as phosphatidyl serine, whereas DnaK interacted with all lipids tested regardless of the charge. Both proteins were inserted into the lipid bilayer as demonstrated by resistance to acid or basic washes, which was confirmed by partial protection from proteolytic cleavage. The majority of HspA1 was inserted into the membrane with a small region of the N-terminus end exposed to the outer phase of the liposome. In contrast, the N-terminus end of DnaK was inserted into the membrane, exposing the C-terminus end outside the liposome. HspA1 was found to make high oligomeric complexes upon insertion into the membranes whereas DnaK only formed dimers within the lipid membrane. These observations suggest that both Hsp70s interact with lipids, but mammalian HspA1 displayed a high degree of specificity and structure as compared with the bacterium form. 2840-Pos Board B217 Role of PIP2-Dependent Membrane Interactions in Vinculin Activation, Motility and Force Transmission Sharon L. Campbell1, Peter M. Thompson1, Caitlin E. Tolbert2, Lindsay Case3, Srinivas Ramachandran1, Mihir Pershad1, Nikolay Dokholyan1, Keith Burridge2, Clare Waterman3. 1 Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA, 2Cell Biology, University of North Carolina, Chapel Hill, NC, USA, 3Cell and Biology Center, NIH; NHLBI, Bethesda, MD, USA. Vinculin is an essential and abundant cytoskeletal protein localized to focal adhesions and cell-cell contacts, where it participates in the linkage of transmembrane receptors to the actin cytoskeleton to control cell survival and migration. Loss of vinculin results in increased cell migration, apoptotic resistance, and the acquisition of tumorigenic properties. Mutations in vinculin and its splice variant, metavinculin, are associated with cardiac disease. Vinculin consists of a head domain (Vh) and a tail domain (Vt) that form autoinhibitory interactions in its inactive state, but release upon activation, exposing phosphorylation, protein and phosphoinositol 4,5-bisphosphate (PIP2) binding sites. The interaction of vinculin with PIP2 is believed critical for its function, however, it is currently unclear how vinculin specifically recognizes PIP2 and regulates vinculin. Vt forms an antiparallel five-helix bundle with amino-terminal (NT) and carboxyl terminal (CT) extensions. While a crystal structure of an oligomerized Vt mutant complexed to a short chain PIP2 has recently been published (Chinthalapudi et al. (JCB, 2014)), the structure is incompatible with membrane insertion. We propose an alternative model using experimental data, molecular docking and dynamics simulations and provide validation of the model through biophysical and biochemical analyses. In our model, the PIP2 head group binds to the Vt basic collar, and promotes release of Vt’s strap and CT to facilitate membrane insertion. The role of vinculin/PIP2 interaction in mediating vinculin activation, localization, cell migration, force sensing and transmission has also been characterized using cell microscopy, including super-resolution microscopy approaches, by examining WT and PIP2-deficient full length vinculin in knockout cells. Information derived from these analyses will result in an unprecedented understanding of vinculin function from the molecular to the cellular level and will enable us to build more comprehensive models of vinculin membrane interactions. 2841-Pos Board B218 Orientation of Dimeric Tubulin on Lipid Membranes Studied using Neutron Reflectometry David P. Hoogerheide1, Sergei Noskov2, Tatiana K. Rostovtseva3, Sergey M. Bezrukov3, Hirsh Nanda1,4. 1 Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD, USA, 2Biochemistry Department, University of Calgary, Calgary, AB, Canada, 3Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA, 4Physics Department, Carnegie Mellon University, Pittsburgh, PA, USA. Dimeric tubulin has emerged as an important regulatory factor of the permeability of voltage-dependent anion channel (VDAC) in the mitochondrial

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outer membrane, with implications for mitochondrial energetics as well as the Warburg effect observed in cancer cells. Previously, single-channel studies revealed that the on-rate of the VDAC-tubulin interaction is strongly dependent on the lipid environment. To understand the orientation of this abundant cytosolic water-soluble protein when bound to lipid membranes, we have employed neutron reflectometry (NR) of tubulin on a tethered bilayer lipid membrane (tBLM) platform. NR is particularly wellsuited to study the profile of high-molecular weight proteins associated with membranes because contrast can be generated with mixtures of light and heavy water, and because of the variation in the cross-section of neutron scattering from the various light elements composing biological materials. The NR data are consistent with two models of tubulin binding, in which the tubulin heterodimer is bound to the membrane surface by either its alpha- or beta-subunit, but not both simultaneously. This result is consistent with coarse-grained molecular dynamics (MD) simulations of the interaction of the alpha subunit of tubulin with membranes of different compositions. The binding surfaces derived from the NR results are highly conserved and correspond to the dimer-dimer interface in microtubule protofilaments. We discuss the implications of the orientation of membrane-bound dimeric tubulin for the mechanism by which tubulin regulates VDAC, including the accessibility of the negatively charged C-terminal tails for VDAC pore blockage and the possibility that the two subunits have different regulatory roles. 2842-Pos Board B219 Shape Transformaiton of Biomembrane Induced by Banana-Shaped Protein Rods: Tubulation and Formation of Polyhedral Vesicles Hiroshi Noguchi. Institute for Solid State Physics, University of Tokyo, Kashiwa, Japan. In living cells, morphology of biomembranes is regulated by various proteins. Many of these proteins contain a banana-shaped binding module called BAR (Bin-Amphiphysin-Rvs) domain. We have studied how anisotropic spontaneous curvatures of banana-shaped protein rod induce effective interaction between the proteins and change membrane shapes by using implicit-solvent meshless membrane simulations [1-3]. We found that the self-assembly of the rods is divided to two directional assemblies at the low rod density [1] and polyhedral vesicles and polygonal tubes are formed at the high density [2]. We also revealed that a small spontaneous curvature perpendicular to the rod can remarkably alter the tubulation dynamics at high rod density whereas minor effects are only obtained at low density [3]. A percolated network, which suppresses tubule protrusion, is intermediately formed for negative perpendicular curvatures. [1] Noguchi, H. (2014) EPL 108, 48001. [2] Noguchi, H. (2015) J. Chem. Phys. 143, 243109. [3] Noguchi, H. arXiv:1503.00973. 2843-Pos Board B220 Introducing Improved Protein Side Chain Dynamics in the MARTINI Model to Simulate Protein-Membrane Interactions Florian A. Herzog, Lukas Braun, Ingmar Schoen, Viola Vogel. ETH Zurich, Zurich, Switzerland. Specific protein-lipid interactions regulate the localization and activity of many proteins. Here, we present a computational approach how to determine the orientation and specific interactions of peripheral proteins at lipid membranes based on an improved version of the coarse-grained MARTINI model. To avoid unphysical side chain orientations that commonly occur in b-strands during MARTINI simulations, we introduce a modification of the MARTINI protein model. This modification - called ProtFix MARTINI - was validated with atomistic molecular dynamics simulations of three different proteins in solution and applied to the membrane binding of PLC-d1 pleckstrin homology (PH) domain. The modification is necessary and sufficient to reproduce the lipid-protein interactions seen in ms long atomistic molecular dynamics simulations of the well-studied PH domain. In contrast to atomistic simulations, ProtFix MARTINI simulations allow for a complete sampling of the rotational diffusion of the protein at the membrane. By running multiple simulations from systematically chosen initial orientations, we were able to characterize the free energy landscape of binding and observed a convergence through several transient states towards one unique binding orientation within the simulated time. In this orientation, PIP2 molecules bound stably to the known binding pocket of the PH domain and transiently to so-far unknown binding sites. Both the unique orientation and the specific protein side chain-lipid interactions are in excellent agreement with results from numerous experimental studies. We therefore propose that the predictive power of the ProtFix MARTINI model can generate experimentally testable hypotheses to finally

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improve on our understanding of the dynamics of protein-membrane interactions at different time scales. 2844-Pos Board B221 Transportation of an Artifical Cargo by a Par-Min Hybrid System James A. Taylor1, Anthony G. Vecchiarelli1, Keir C. Neuman2, Kiyoshi Mizuuchi1. 1 LMB, NIDDK, Bethesda, MD, USA, 2LSMB, NHLBI, Bethesda, MD, USA. Spatial organization and active transportation of organelles are essential processes for cellular division. This is true even in bacteria, which were once thought too simple to require much internal organization. The ubiquitous deviant Walker ATPase family of proteins has been shown to be involved in systems important for spatial coordination in bacteria, the two most highly studied examples of which are the DNA partitioning Par system and the divisome positioning Min system. Both these systems contain the eponymous deviant Walker A ATPase (ParA or MinD), a protein which stimulates the ATPase’s hydrolysis activity (ParB or MinE) and a cargo to be transported or localized (a ‘‘centromere’’ DNA site bound by ParB or the divisome inhibitor MinC bound to MinD). Despite the similarities of their components these systems appear to behave quite differently in vivo: the Par system segregates large clusters of ParB bound DNA cargo into sister cells during division by displacing ParA non-specifically bound to the nucleoid whereas the Min proteins bind the inner membrane and are seen to oscillate from one cell pole to the other. These differences may arise due to the nature of the binding surface (DNA vs lipid) or the nature of the cargo (a large cargo bound a large number of stimulator protein molecules in Par vs small protein molecules individually bound to membrane-bound MinD dimers). Here we use TIRF microscopy to demonstrate that the Min system is capable of transporting a magnetic bead (a large cargo) along a supported lipid bilayer in a manner strikingly similar to our previous reconstitution of the Par system. This demonstrates that despite their clear differences in their system dynamics under conditions for their natural functions, Par and Min systems operate based on common underlying mechanistic principles. 2845-Pos Board B222 New Insights on the Lipidation of Peptides and Proteins in Lipid Membranes Hannah M. Britt, Vian S. Ismail, John M. Sanderson. Dept. of Chemistry, Durham University, Durham, United Kingdom. Peptides have been shown to undergo intrinsic lipidation reactions in lipid membranes (http://dx.doi.org/10.1016/j.jmb.2013.07.013), involving acyl transfer from lipid constituents of the membrane to acceptor sites on the peptide. The physical properties of the membrane that promote this process have been investigated and are rationalized in terms of the rate determining step in the process and the curvature modulus of the membrane. The lipidation by-products, lyso-lipids, may also serve as acyl group donors for further lipidation. Intrinsic lipidation is not restricted to peptides: proteins and small molecules also undergo the process. We have examined the lipidation profiles of aquaporin-0 and shown that it can be accounted for with knowledge of the fatty acyl composition of lens membranes, thereby supporting the hypothesis that their lipidation arises from membrane lipid precursors. Current work to understand the factors that dictate this reactivity, and the consequences of lipidation, will be presented. 2846-Pos Board B223 A Novel Experimental Platform to Study the Temporal Evolution of Phosphoinositide Gradients in Model Membranes Brittany M. Neumann1,2, Devin Kenney3, Qi Wen4, Arne Gericke1. 1 Chemistry Biochemistry, Worcester Polytechnic, Worcester, MA, USA, 2 Chemistry Biochemistry, Worcester Polytechnic Institute, Worcester, MA, USA, 3Chemistry Biochemistry, BridgeWater State University, Bridgewater, MA, USA, 4Physics, Worcester Polytechnic, Worcester, MA, USA. A broad range of cellular functions have been linked to dynamically controlled phosphoinositide gradients, including the migration of Dictyostelium discoideum and human neutrophils. For efficient Dictyostelium migration phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) gradients are maintained through positive Ras/PI(3,4,5)P3 and negative Ras/TorC2/Akt feedback loops. Currently in the field there are several mathematical models that have been proposed to describe the dynamics of these phosphoinositide gradients. However, there are exceedingly few methods to test and expand these hypotheses using real model membranes with defined lipid and protein compositions of varying lateral heterogeneities. We have developed a novel experimental platform that takes advantage of the lamellar flow of microfluidics to study phos-

phoinositide gradients in supported lipid bilayers (SBL). With this platform we have successfully generated the phosphoinositide gradients in SBL. We quantified the gradients by total internal reflectance fluorescence microscopy (TIRF) and studied how these gradients are affected by external factors such as calcium. In addition, this platform can also be used to study the impact of proteins on the lipid gradient. We studied proteins such as the tumor suppressor protein PTEN that dephorphorylates PI(3,4,5)P3 to PI(4,5)P2 and how this enzyme can remodel the SBL in a gradient dependent manner. This novel platform can inform how the association of these proteins can impact such lipid gradients and begin to unravel how microorganisms maintain their complex lateral membrane organization. 2847-Pos Board B224 Dynamic Contacts between the Endoplasmic Reticulum and the Plasma Membrane Regulate Phosphoinositide Metabolism and Control Cellular Excitability Eamonn J. Dickson, Jill B. Jensen, Bertil Hille. Physiology and Biophysics, Univeristy of Washington, Seattle, WA, USA. Contacts between the endoplasmic reticulum and the plasma membrane (ERPM junctions) facilitate intracellular communication and are essential to excitation-contraction coupling and store-operated calcium entry (SOCE). We find that they also regulate membrane phosphoinositides, lipids that control ion channels and electrical excitability. We examined how Sac1, an ER-integral 4-phosphatase, regulates PM phosphoinositides via ER-PM junctions. Through the use of HPLC mass spectrometry to quantitatively monitor phosphoinositides, and super resolution microscopy to determine the localization of Sac1, we have discovered that at steady state the cortical ER tethering protein extended synaptotagmin 2 (E-Syt2) positions the ER and Sac1 in discrete ER-PM junctions. Here, the enzyme’s phosphatase activity participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2. Activation of G-protein coupled receptors that deplete PM PI(4,5)P2 also dynamically redistributes the ER, and as a consequence Sac1, away from the PM. Conversely, physiological depletion of ER luminal calcium and consequent activation of SOCE leads to increased contact between the ER and the PM and enhanced Sac1 depletion of PM PI(4)P. Further, Sac1 overexpression increases the number of STIM1 puncta at steady-state and during SOCE, resulting in higher cytoplasm calcium levels at rest and during calcium entry. Thus, the dynamic presence of Sac1 at ER-PM junctions allows it to act as a cellular sensor and controller of PM phosphoinositides and thereby influence many PM processes including electrical excitability. Supporting NIH grant R37NS08174. 2848-Pos Board B225 Endolysin PlyC Binding to Model Membranes Reveals its Entry Point on Mammalian Cells Marilia Barros1,2, Tarek Vennemann1, Frank Heinrich1,2, Daniel Nelson3, Mathias Lo¨sche1,2. 1 Department of Physics, Carnegie Mellon University, Pittsburgh, PA, USA, 2 NIST Center for Neutron Research, Gaithersburg, MD, USA, 3Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA. Endolysins are bacteriophage-encoded peptidoglycan hydrolases expressed during the late stages of a phage replication cycle. that function to lyse the bacterial cell wall, thus enabling progeny phage release. When exogenously added, these enzymes lyse the peptidoglycan of Gram-positive pathogens, resulting in osmotic lysis and cell death. One particular endolysin, PlyC, gains cellular entry and clears intracellular streptococci without compromising cell viability, implying that an unknown mechanism provides entry to the streptococcispecific endolysin into mammalian cells. Here, we investigate the molecular basis of PlyC binding domain, PlyCB, ability to bind and cross the mammalian plasma membrane using synthetic model membranes. Complementary surface-sensitive techniques showed membrane integrity during PlyCB exposure and quantified membrane-binding affinity. Surface plasmon resonance data reveals that while the interaction of PlyCB with purely zwitterionic membranes is negligible, the protein strongly interacts with anionic membranes that contain phosphatidylserine (PS) above a welldefined concentration threshold. In contrast, PlyCB affinity for other anionic lipids tested is low, suggesting specificity for PS. Neutron reflection data showed that PlyCB binding to the membrane surface is followed by penetration into the hydrophobic membrane core, while impedance spectroscopy confirms that the membrane integrity is not affected. Those findings provide clues to a mechanistic understanding of how PlyCB binds and initiates membrane translocation. After the initial membrane interaction, PlyCB is known to