Quantifying the Ability of Clathrin Triskelia to Sense Membrane Curvature

Wednesday, February 15, 2017 normally cleared from the brain before it exerts any apparent toxicity. Under some conditions, however, it undergoes a conformational change and aggregates into fibrils with cross-beta structure. These fibrils then coalesce into amyloid plaques,which are the pathognomonic brain lesions of Alzheimer’s disease. The plaques are centers of active oxidative stress and neuronal death, so the conditions under which the conformational change of Ab is of high interest. When Ab is encapsulated in a reverse micelle under laboratory conditions, infrared spectroscopy indicates that it spontaneous adopts an extended beta-sheet conformation, which is remarkable because only one Ab strand is present in each reverse micelle. That observation suggests that some aspect of the reverse micelle environment such as crowding, dehydration, proximity to a membrane, or high ionic strength can induce Ab to nucleate amyloid fibril formation. Therefore, an understanding of the factors that induce Ab to adopt this conformation in reverse micelles may reveal how it is induced to form amyloid fibrils in Alzheimer’s disease. ms lond Molecular dynamics simulations of Ab in reverse micelles have been performed on supercomputer Anton to identify and understand these factors. Results indicate that Ab side chain interactions with the reverse micelle surface help stabilize intrachain hydrogen bond formation and secondary structure formation. These results also represent important sequence-specific details within an increasingly large body of evidence suggesting that Ab-membrane interactions are important for the formation of amyloid fibrils in Alzheimer’s disease. 2609-Pos Board B216 Lipid-Peptide Interaction Dynamics with Reaction-Diffusion Fluorescence Correlation Spectroscopy Xiaosi Li, Xiaojun Shi, Adam W. Smith. University of Akron, Chem Dept, Akron, OH, USA. Peptides containing positively charged residues interact with anionic lipids in the plasma membrane. Phosphatidylinositol 4,5-bisphophate (PIP2), an important anionic lipid in the cytoplasmic leaflet of the plasma membrane, is recruited to membrane-associated proteins that contain basic domains. Despite the biological importance of PIP2 lipids and peripheral proteins in cell signaling, we know little about their affinity and interaction kinetics. Here, we used pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to assess the mobility and correlated diffusion of two peripheral peptides with PIP2 lipids on asymmetric supported lipid bilayers. Two histidine-tagged peptides containing basic residues were designed to investigate this electrostatic interaction, the first peptide had eight lysine residues (His8-Lys8) and the second one corresponding to the basic domain of protein MARCKS (His8-MARCKS(152-176)). We use a reaction-diffusion model to analyze the FCS data, from which we are able to extract lipid-peptide association and dissociation rates. Our results provide direct evidence for the formation of a stable peptide-lipid complex. The experimentally determined equilibrium constant reports on the binding energy of PIP2 with the His8Lys8 and His8-MARCKS(152-176) peptides. By altering the buffer conditions, the binding affinity and kinetics was modified for each peptide. Overall, our results provide unique insight into the dynamics of lipid-peptide interactions. 2610-Pos Board B217 Apelin and Apela, Ligands for the Same GPCR, Differ in their Isoformand Headgroup-Dependent Micelle Interaction Kyungsoo Shin, Muzaddid Sarker, Shuya K. Huang, Jan K. Rainey. Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS, Canada. The membrane catalysis theory states that ligands associate with membranes to enhance its rate of binding to cell surface receptors. The initial association increases local concentration of ligand, reduces diffusion from a 3D to a 2D process, and/or induces conformational change for receptor recognition. Variations in membrane composition mean that ligands may encounter a variety of environments, with potential for lipid-dependent preferences in both binding and conformation. We tested for evidence of membrane catalysis with apelin and apela, two peptide hormones of a single Class A GPCR (the apelin receptor). Both hormones can be processed into multiple isoforms. Apelin exists as 55, 36, 17, or 13-residue isoforms, apela as 32, 22, or 11-residue isoforms. All isoforms retain the C-terminal residues to bind to and activate the receptor, in turn regulating a variety of physiological systems. Far-UV CD and solution-state NMR spectroscopy demonstrate that all apelin isoforms exhibit b-turn characteristics in the presence of anionic, but not zwitterionic, micelles, suggestive of a preferential lipid interaction. Conversely, apela-32 exhibited a similar level of conformational change with both zwitterionic and anionic micelles, but removal of the N-terminal region led to disproportionate level of micellemediated changes, as observed in apela-11. Thus, membrane-association affects apelin and apela isoforms differently in response to membrane composition although they are ligands of a single GPCR. Since composition of cell and

529a

organelle membranes can vary, preferential membrane-ligand association may regulate the rate of apelin/apela-GPCR binding (potency & efficacy), signaling pathways (Gai vs. b-arrestin), and signaling mechanisms (endocrine vs. autocrine). Characterizing ligands of the apelin receptor presents a rare opportunity to test for membrane catalysis as a method to control and diversify hormonal signaling mechanisms both in physiological conditions and for therapeutic targeting. 2611-Pos Board B218 Molecular Basis of Ligand Binding by the Endosomal Adaptor Protein Tom1 Wen Xiong1, Ji Woong Choi1, Xiaolin Zhao1, Jeff F. Ellena2, Daniel G.S. Capelluto1. 1 Protein Signaling Domains Laboratory, Department of Biological Sciences, Biocomplexity Institute, Virginia Tech, Blacksburg, VA, USA, 2Department of Chemistry, University of Virginia, Charlottesville, VA, USA. Tom1 (target of Myb 1) plays a role in membrane trafficking by serving as an alternative endosomal sorting complex required for transport (ESCRT)0 component. Tom1 possesses an N-terminal VHS domain followed by a central GAT domain. Tom1 has been shown to serve as a new phosphatidylinositol 5-phosphate (PI(5)P) effector at signaling endosomes through its VHS domain, delaying cargo degradation in a bacterial infection model. The Tom1 VHS domain also binds ubiquitin moieties in cargo for endosomal transport and degradation; therefore, we hypothesize that the ubiquitin and PI(5)P compete each other for Tom1 VHS binding. In order to address this question, the backbone NMR resonances of Tom1 VHS were assigned. The Tom1 VHS secondary structure prediction scores, using TALOSþ, are in good agreement with the secondary structural elements reported for the crystal structure of the protein. Our heteronuclear single quantum coherence data revealed that Tom1 VHS interacts with PI(5)P following a fastexchange regime, with the PI(5)P binding site predicted to be at a region spanning a-helices 6 and 8. In contrast, we found that the ubiquitinbinding site in Tom1 VHS is located at the a-helices 2, 5 and 7 of Tom1 VHS. Despite the binding sites are not overlapped, the ubiquitin and PI(5) P may compete each other by inducing conformational changes in the Tom1 VHS domain upon binding. Also, we identified a conserved central hydrophobic patch at the ubiquitin surface to be the binding site for the Tom1 VHS domain. The ubiquitin hydrophobic patch is also involved in Tom1 GAT domain binding, suggesting that Tom1 can bind ubiquitin molecules through two independent sites. By providing the molecular basis of the Tom1 interactions, we will generate cargo sorting mechanistic insights, create functionally specific mutations, and precisely manipulate alternative ESCRT-0 proteins. 2612-Pos Board B219 Quantifying the Ability of Clathrin Triskelia to Sense Membrane Curvature Avinash Gadok, Jeanne Stachowiak. University of Texas, Austin, TX, USA. Clathrin-mediated endocytosis is a primary pathway of entry into the cell. Therefore, understanding the molecular mechanisms that drive the assembly of the clathrin coat is a fundamental physical problem in biology. Adaptor proteins, which bind to the membrane and to clathrin, are responsible for the biochemical recruitment of clathrin to endocytic structures. However, the physical cues that influence clathrin’s recruitment remain debated. In particular, while adaptor proteins such as epsin and amphiphysin bind preferentially to regions of high membrane curvature, it remains unknown whether clathrin triskelia themselves possess the ability to sense membrane curvature and bind preferentially to curved structures. To address this question, we isolated clathrin’s curvature sensing ability in the absence of adaptor proteins by using recombinant, histidine-tagged clathrin that is engineered to bind directly to Ni-NTA-containing membranes. To examine clathrin binding to these membranes, we used a quantitative fluorescence intensity-based approach and quantified the bound triskelia to lipid ratio for individual vesicles spanning a broad range of diameters. Our results demonstrate that clathrin binds preferentially to membranes of higher curvature. Specifically, when incubated with the same concentration of clathrin triskelia, vesicles with an average diameter of 30 nm recruit twice as many clathrin triskelia per membrane surface area in comparison to vesicles with an average diameter of 400 nm. Our ongoing work is mapping the distribution of bound triskelia over vesicles with a range of different curvatures. This data is being used to make a statistical model from which the curvature dependence of clathrin-lipid binding energy can be derived. These findings will provide fundamental insight into the process by which clathrin is recruited to highly curved membranes during clathrin-mediated endocytosis.