- Email: [email protected]
Human and Bacterium Hsp70 Interacts Differently with Lipid Membranes
574a
Wednesday, March 2, 2016
in response to QPVP and MARCKS binding was evaluated by a time-resolved fluorescence technique, pulsed interleaved excitation fluorescence crosscorrelation spectroscopy (PIE-FCCS), along with single particle tracking (SPT) of individual PIP2 molecules. The trajectories of individual PIP2 lipids indicate intermittent-type diffusion in which confined small steps are separated by steps typical of free Brownian motion. The confined movement is a result of stalling during binding events with QPVP molecules and reveals the microscopic details of the PIP2 lipid mobility decrease observed previously by FCCS measurements. FCCS measurements also indicate a collective diffusion of a cationic peptide, MARCKS, and PIP2, suggesting the existence of a large peptide-lipid assembly formed through electrostatic sequestration of PIP2 lipids by MARCKS. SPT analysis shows the diffusive behavior the peptide-lipid assemblies. The results suggest that PIP2 diffusion is significantly altered upon binding to polycationic molecules and is the first direct experimental evidence of stable lipid-peptide complexes formed through electrostatic interactions between MARCKS peptide and PIP2 lipid. 2835-Pos Board B212 Membrane Fission by Protein Crowding Wilton T. Snead, Carl C. Hayden, Jeanne C. Stachowiak. Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA. Understanding the molecular mechanisms that underlie membrane fission is a key physical problem in biology. The epsin1 N-terminal homology (ENTH) domain is a potent driver of membrane curvature, and has recently been shown to play a role in membrane fission. Specifically, a recent report proposed that insertion of a wedge-like amphipathic helix by ENTH curves and destabilizes membranes, as evidenced by decreasing membrane fission ability among ENTH mutants with decreasing helix hydrophobicity (Boucrot et al., Cell 2012). However, our group recently showed that collisions among dense, membrane-bound ENTH proteins generate steric pressure, which drives membrane bending in the absence of helix insertions (Stachowiak et al., Nature Cell Biology 2012). These results prompted us to ask: is steric pressure also responsible for membrane fission by ENTH? To answer this question, we correlate lifetime FRET measurements of the surface density of membrane-bound proteins with quantitative membrane fission measurements. We find that ENTH mutants with reduced helix hydrophobicity also have dramatically reduced membrane affinity. However, these mutants are capable of driving fission to a similar degree as wild-type ENTH when bound to the membrane at comparable density. Interestingly, we find that full-length epsin, which contains a bulky, intrinsicallydisordered C-terminal domain, drives fission more potently than ENTH alone. Our results imply that, while helix insertions are important for binding proteins tightly to membrane surfaces, helices are not required for fission. However, once bound to the membrane surface at sufficient density, bulky molecules of arbitrary structure can create steric pressure that increases membrane curvature until fission occurs spontaneously. Taken together, our findings reveal a novel, non-specific mechanism for membrane fission, which may work in concert with the structure-mediated specificity of fission machines like dynamin, in order to ensure robust membrane fission in diverse biological contexts. 2836-Pos Board B213 Understanding the Role of Peptide-Lipid Reactions in Biological Systems Hannah M. Britt, Vian S. Ismail, Jackie A. Mosely, John M. Sanderson. Department of Chemistry, Durham University, Durham, United Kingdom. The field of reactions in lipid membranes, so called intra-membrane reactions, has thus far been limited by the sensitivity and availability of analytical techniques suitable for their study. Use of LCMS with a high performance FTICR mass analyser has opened up this field of complex reactions dramatically, revealing in particular the existence of a novel intra-membrane reaction, intrinsic lipidation. Intrinsic lipidation describes transfer of the acyl chain of a membrane phospholipid onto the nucleophile of a membrane active peptide, such as melittin. Unlike post-translational peptide lipidation, intrinsic lipidation in the membrane proceeds without the aid of an enzyme. Identified as a process generic to membrane active peptides in vitro, intrinsic lipidation has been implicated in several antimicrobial and disease-related contexts, not least as a potential mechanism for amyloid plaque formation in Alzheimer’s and other amyloid-related degenerative diseases. Further, promising early results suggest intrinsic lipidation takes place in vivo, playing a role in the modification of natural protein aquaporin-0. Whilst the impact of lipidation on this protein is currently poorly understood, it does signify a potentially novel means of drug targeting. Focus is now upon conclusively confirming the role of intrinsic lipidation in both natural systems and in disease-related con-
texts. Further, by probing factors associated with the reactivity of intrinsic lipidation it is hoped that a heightened understanding of the reaction will be reached, allowing manipulation of the reaction for drug design and disease prevention. Our most recent data on peptide and protein lipidation are presented. 2837-Pos Board B214 Selective Targeting of Lipid Droplets by Proteins Morris E. Cohen, Gregory A. Voth. Department of Chemistry, University of Chicago, Chicago, IL, USA. Lipid droplets (LDs) are organelles that store neutral lipids in cells. LDs have a core of neutral lipids, including triacylglycerols and sterol esters, surrounded by a phospholipid monolayer. LDs can grow dramatically to store neutral lipids in its core, which requires a commensurate increase in the size of the LD membrane. CTP:phosphocholine cytidylyltransferase (CCT) plays a key role in regulating the size of LDs, as it the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, which is a major component of the LD outer coat. CCT has been shown to have a binding preference for LD as opposed to bilayer membranes, although the cause of this preference is not known. Previous studies have shown that large hydrophobic packing defects are key for the binding of amphipathic helices (AH) to membranes. We have studied LDs and bilayer membranes by extensive long time scale molecular dynamics (MD) simulations. In our results, we observe differing properties between LD membranes and bilayer membranes, including an increase in the frequency of large defects in LD membranes. We also find, using ms-long MD simulations, that an increase in the size of hydrophobic lipid packing defects in LDs compared to bilayer membranes plays a large role in the specificity of CCT AH initial binding to membranes as compared to bilayer membranes. Moreover, we frequently observe detachment of CCT from bilayer membranes, suggesting the lack of large hydrophobic defects destabilizes this interaction sufficiently to make binding to bilayers thermodynamically less favorable. 2838-Pos Board B215 Membrane Lateral Pressure Controls Hydration and Water Mobility at the Copper-Binding Site of the P1B-type Copper ATPase CopA from Legionella Pneumophila Karim Fahmy, Elisabeth Fischermeier, Ahmed Sayed. Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany. P-type ATPases couple ATP hydrolysis to ion transport. We have reconstituted the copper-transporting P1B-type ATPase LpCopA from Legionella pneumophila into lipid nanodiscs in order to study the influence of membrane lateral pressure on the functionally relevant intra-membrane protein hydration at the ion-binding site. Using site-directed mutagenesis, the solvatochromic fluorophore BADAN was covalently linked to the cysteine residues at the conserved copper-binding CPC motif on transmembrane helix 4. The decomposition of the fluorescence spectra of labeled LpCopA in the micellar and the lipid-inserted state shows that membrane lateral pressure reduces hydration and water mobility in the environment of the more buried Cys-382 with a concomitant change of the local dielectric constant by 9. In contrast, the environment of Cys-384 which is located closer to the putative membrane surface, resembles a ‘‘hydrophobic gate’’ with low water mobility that is little affected by insertion into a bilayer (change of local dielectric constant by 3). The asymmetric hydration and water mobility around the CPC motif provides Cys-382 with a highly dynamic hydration. The data show that membrane lateral pressure may provide a restoring force in hydration / dehydration cycles around Cys-382 in the transmembrane domain during catalytic activity. The lower hydration and mobility in the Cys-384 environment, on the other hand, would favor the dehydration of copper and hinder its re-solvation from the intracellular side. 2839-Pos Board B216 Human and Bacterium Hsp70 Interacts Differently with Lipid Membranes Victor Lopez1, David M. Cauvi2, Nelson Arispe3, Antonio De Maio4. 1 Initiative for Maximizing Student Development (IMSD) Program, University of California, San Diego, La Jolla, CA, USA, 2Department of Surgery, University of California, San Diego, La Jolla, CA, USA, 3 Department of Anatomy, Physiology and Genetics, Uniformed Services University, Bethesda, MD, USA, 4Department of Surgery and Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, CA, USA. The cellular response to stress is orchestrated by the expression of a family of proteins coined heat shock proteins (hsp), which are involved in the stabilization of basic cellular processes to preserve cell viability and homeostasis.
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
575a
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
Wednesday, March 2, 2016
in response to QPVP and MARCKS binding was evaluated by a time-resolved fluorescence technique, pulsed interleaved excitation fluorescence crosscorrelation spectroscopy (PIE-FCCS), along with single particle tracking (SPT) of individual PIP2 molecules. The trajectories of individual PIP2 lipids indicate intermittent-type diffusion in which confined small steps are separated by steps typical of free Brownian motion. The confined movement is a result of stalling during binding events with QPVP molecules and reveals the microscopic details of the PIP2 lipid mobility decrease observed previously by FCCS measurements. FCCS measurements also indicate a collective diffusion of a cationic peptide, MARCKS, and PIP2, suggesting the existence of a large peptide-lipid assembly formed through electrostatic sequestration of PIP2 lipids by MARCKS. SPT analysis shows the diffusive behavior the peptide-lipid assemblies. The results suggest that PIP2 diffusion is significantly altered upon binding to polycationic molecules and is the first direct experimental evidence of stable lipid-peptide complexes formed through electrostatic interactions between MARCKS peptide and PIP2 lipid. 2835-Pos Board B212 Membrane Fission by Protein Crowding Wilton T. Snead, Carl C. Hayden, Jeanne C. Stachowiak. Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA. Understanding the molecular mechanisms that underlie membrane fission is a key physical problem in biology. The epsin1 N-terminal homology (ENTH) domain is a potent driver of membrane curvature, and has recently been shown to play a role in membrane fission. Specifically, a recent report proposed that insertion of a wedge-like amphipathic helix by ENTH curves and destabilizes membranes, as evidenced by decreasing membrane fission ability among ENTH mutants with decreasing helix hydrophobicity (Boucrot et al., Cell 2012). However, our group recently showed that collisions among dense, membrane-bound ENTH proteins generate steric pressure, which drives membrane bending in the absence of helix insertions (Stachowiak et al., Nature Cell Biology 2012). These results prompted us to ask: is steric pressure also responsible for membrane fission by ENTH? To answer this question, we correlate lifetime FRET measurements of the surface density of membrane-bound proteins with quantitative membrane fission measurements. We find that ENTH mutants with reduced helix hydrophobicity also have dramatically reduced membrane affinity. However, these mutants are capable of driving fission to a similar degree as wild-type ENTH when bound to the membrane at comparable density. Interestingly, we find that full-length epsin, which contains a bulky, intrinsicallydisordered C-terminal domain, drives fission more potently than ENTH alone. Our results imply that, while helix insertions are important for binding proteins tightly to membrane surfaces, helices are not required for fission. However, once bound to the membrane surface at sufficient density, bulky molecules of arbitrary structure can create steric pressure that increases membrane curvature until fission occurs spontaneously. Taken together, our findings reveal a novel, non-specific mechanism for membrane fission, which may work in concert with the structure-mediated specificity of fission machines like dynamin, in order to ensure robust membrane fission in diverse biological contexts. 2836-Pos Board B213 Understanding the Role of Peptide-Lipid Reactions in Biological Systems Hannah M. Britt, Vian S. Ismail, Jackie A. Mosely, John M. Sanderson. Department of Chemistry, Durham University, Durham, United Kingdom. The field of reactions in lipid membranes, so called intra-membrane reactions, has thus far been limited by the sensitivity and availability of analytical techniques suitable for their study. Use of LCMS with a high performance FTICR mass analyser has opened up this field of complex reactions dramatically, revealing in particular the existence of a novel intra-membrane reaction, intrinsic lipidation. Intrinsic lipidation describes transfer of the acyl chain of a membrane phospholipid onto the nucleophile of a membrane active peptide, such as melittin. Unlike post-translational peptide lipidation, intrinsic lipidation in the membrane proceeds without the aid of an enzyme. Identified as a process generic to membrane active peptides in vitro, intrinsic lipidation has been implicated in several antimicrobial and disease-related contexts, not least as a potential mechanism for amyloid plaque formation in Alzheimer’s and other amyloid-related degenerative diseases. Further, promising early results suggest intrinsic lipidation takes place in vivo, playing a role in the modification of natural protein aquaporin-0. Whilst the impact of lipidation on this protein is currently poorly understood, it does signify a potentially novel means of drug targeting. Focus is now upon conclusively confirming the role of intrinsic lipidation in both natural systems and in disease-related con-
texts. Further, by probing factors associated with the reactivity of intrinsic lipidation it is hoped that a heightened understanding of the reaction will be reached, allowing manipulation of the reaction for drug design and disease prevention. Our most recent data on peptide and protein lipidation are presented. 2837-Pos Board B214 Selective Targeting of Lipid Droplets by Proteins Morris E. Cohen, Gregory A. Voth. Department of Chemistry, University of Chicago, Chicago, IL, USA. Lipid droplets (LDs) are organelles that store neutral lipids in cells. LDs have a core of neutral lipids, including triacylglycerols and sterol esters, surrounded by a phospholipid monolayer. LDs can grow dramatically to store neutral lipids in its core, which requires a commensurate increase in the size of the LD membrane. CTP:phosphocholine cytidylyltransferase (CCT) plays a key role in regulating the size of LDs, as it the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, which is a major component of the LD outer coat. CCT has been shown to have a binding preference for LD as opposed to bilayer membranes, although the cause of this preference is not known. Previous studies have shown that large hydrophobic packing defects are key for the binding of amphipathic helices (AH) to membranes. We have studied LDs and bilayer membranes by extensive long time scale molecular dynamics (MD) simulations. In our results, we observe differing properties between LD membranes and bilayer membranes, including an increase in the frequency of large defects in LD membranes. We also find, using ms-long MD simulations, that an increase in the size of hydrophobic lipid packing defects in LDs compared to bilayer membranes plays a large role in the specificity of CCT AH initial binding to membranes as compared to bilayer membranes. Moreover, we frequently observe detachment of CCT from bilayer membranes, suggesting the lack of large hydrophobic defects destabilizes this interaction sufficiently to make binding to bilayers thermodynamically less favorable. 2838-Pos Board B215 Membrane Lateral Pressure Controls Hydration and Water Mobility at the Copper-Binding Site of the P1B-type Copper ATPase CopA from Legionella Pneumophila Karim Fahmy, Elisabeth Fischermeier, Ahmed Sayed. Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany. P-type ATPases couple ATP hydrolysis to ion transport. We have reconstituted the copper-transporting P1B-type ATPase LpCopA from Legionella pneumophila into lipid nanodiscs in order to study the influence of membrane lateral pressure on the functionally relevant intra-membrane protein hydration at the ion-binding site. Using site-directed mutagenesis, the solvatochromic fluorophore BADAN was covalently linked to the cysteine residues at the conserved copper-binding CPC motif on transmembrane helix 4. The decomposition of the fluorescence spectra of labeled LpCopA in the micellar and the lipid-inserted state shows that membrane lateral pressure reduces hydration and water mobility in the environment of the more buried Cys-382 with a concomitant change of the local dielectric constant by 9. In contrast, the environment of Cys-384 which is located closer to the putative membrane surface, resembles a ‘‘hydrophobic gate’’ with low water mobility that is little affected by insertion into a bilayer (change of local dielectric constant by 3). The asymmetric hydration and water mobility around the CPC motif provides Cys-382 with a highly dynamic hydration. The data show that membrane lateral pressure may provide a restoring force in hydration / dehydration cycles around Cys-382 in the transmembrane domain during catalytic activity. The lower hydration and mobility in the Cys-384 environment, on the other hand, would favor the dehydration of copper and hinder its re-solvation from the intracellular side. 2839-Pos Board B216 Human and Bacterium Hsp70 Interacts Differently with Lipid Membranes Victor Lopez1, David M. Cauvi2, Nelson Arispe3, Antonio De Maio4. 1 Initiative for Maximizing Student Development (IMSD) Program, University of California, San Diego, La Jolla, CA, USA, 2Department of Surgery, University of California, San Diego, La Jolla, CA, USA, 3 Department of Anatomy, Physiology and Genetics, Uniformed Services University, Bethesda, MD, USA, 4Department of Surgery and Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, CA, USA. The cellular response to stress is orchestrated by the expression of a family of proteins coined heat shock proteins (hsp), which are involved in the stabilization of basic cellular processes to preserve cell viability and homeostasis.
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
575a
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