Biophysics of Lipid-Modified Gtpases Monday, Februa

Monday, February 19, 2018 185a Symposium: Fibril Assembly and Structure: 935-Symp Fibril Formation by Amyloid-beta and by Low-complexity Sequences: In- Progress and Challenges sights from Solid State NMR Robert Tycko. 932-Symp Laboratory of Chemical Physics, NIDDK, National Institutes of Health, High Resolution Structure Determination of Amyloid Fibrils Bethesda, MD, USA. Robert G. Griffin. I will describe results from two projects in which my lab uses solid state NMR Chemistry/Francis Bitter Magnet Laboratory, Massachusetts Institute of methods (plus other techniques) to characterize the molecular structures of am- Technology, Cambridge, MA, USA. yloid fibrils. First, I will review our work on fibrils formed by amyloid-b pep- Many peptides and proteins form amyloid fibrils whose detailed molecular tides. This is a long-running project in which we have shown that amyloid-b structure is of considerable functional and/or pathological importance. fibrils are typically polymorphic at the molecular level, identified certain mo- In this presentation we review the macroscopic structural properties of lecular structural features that distinguish polymorphs from one another, devel- fibrils and outline approaches to determining the microscopic structure oped full molecular structural models for fibrils formed in vitro and fibrils of these systems to high resolution using magic angle spinning (MAS) derived from human brain tissue, and investigated correlations between varia- NMR techniques. In particular, we discuss a series of 2D and 3D heteronu- tions in amyloid-b fibril structure and variations in Alzheimer’s disease charac- clear and homonuclear dipole recoupling experiments involving spectral teristics. Second, I will discuss studies of fibrils formed by the low-complexity assignments and distance and torsion angle measurements aimed at accom- domain of the FUS protein (FUS-LC). This is a collaborative project with the plishing this goal. Key to obtaining high resolution is the ability to measure lab of Steven McKnight at the University of Texas Southwestern Medical Cen- a sufficient number of structural constraints per residue. We discuss the ter, in which we seek to understand the phenomenon of aggregation and fibril structures of three different systems determined using these approaches: formation by low-complexity sequences that are rich in glycine and serine res- (1) a fibril performed by an 11 amino acid peptide derived from transthyr- idues but nearly devoid of hydrophobic residues. From solid state NMR data, etin using a set of 70 torsion angle and distance constraints; (2) fibrils we find (surprisingly) that a specific 57-residue segment constitutes the struc- formed by A-beta (1-42), the toxic species in Alzheimers, using a set of turally ordered, immobilized core of fibrils formed by the 214-residue FUS- >500 distance constraints; and (3) a preliminary structure of fibrils forned LC domain, although the core-forming segment has nearly the same amino by beta-2-microglobulin, the 99 amino acid protein associated with dialysis acid composition as the full FUS-LC sequence. Experiments in the McKnight related amylosis. The spectra also provide information on the arrangement lab indicate a close connection between the FUS-LC fibril core structure and of the monomers in the strands that form sheets, and the sheets that ulti- intermolecular interactions in the phase-separated droplet state of FUS-LC mately form the fibrils. Contrary to conventional wisdom, the spectral solutions. data indicate that the molecules in the fibril are microscopically well ordered. Symposium: Biophysics of Lipid-modified 933-Symp GTPases Aggregation of the TAU Protein: Insights from Atomistic and Mesoscale Simulations 936-Symp Joan-Emma Shea. Ras-Membrane Interactions and their Modulation by Effector Proteins UCSB, Santa Barbara, CA, USA. Roland Winter. In this talk, I will discuss the self-assembly of peptides derived from the Tau TU Dortmund University, Dortmund, Germany. protein. Using atomistic simulations, I will present mechanistic insights into In a combined chemical-biological and biophysical approach we explored the the early stages of fibrillization, and the effect of external agents, such as os- free energy contributions to the membrane partitioning of Ras proteins, such as molytes, in either promoting or disfavoring aggregation. I will also present N-Ras and K-Ras4B, and compared the theoretical predictions with experi- field-theoretic simulation of the assembly of Tau peptides into coacervate mental data on the membrane insertion of Ras proteins of various anchor sys- structures. tems into rationally designed model and natural membrane systems. Various factors fostering or reducing the membrane partitioning properties are dis- 934-Symp cussed, including hydrophobic effects, lipid chain mismatch, electrostatic inter- b High Resolution Fibril Structure of Amyloid- (1-42) by Cryoelectron actions, membrane-mediated protein-protein interactions, and terms that Microscopy account for line tension effects between coexisting lipid domains and lipid sort- Dieter Willbold1,2. ing, and change the lateral organization of the lipid bilayer system. For hetero- 1 2 € ICS-6, Reserch Centre Juelich, Julich, Germany, Institut fur Physikalische geneous membranes, localization and sequestration at domain boundaries as € € € Biologie, Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Germany. well as formation of protein clusters and collective lateral organization via b We present the structure of an A (1-42) fibril composed of two intertwined an effective lipid sorting mechanism provide complementary ways of inducing protofilaments determined by cryoelectron microscopy (cryo-EM) to 4.0- membrane nanodomains that could potentially operate as effective signaling ˚ Angstrom resolution, complemented by solid-state nuclear magnetic reso- platforms. PDEd and the Ca-binding protein calmodulin (CaM) are known to nance experiments. The backbone of all 42 residues and nearly all side function as potential binding partners for farnesylated Ras proteins, leading chains are well resolved in the EM density map, including the entire N ter- to a modulation of the spatiotemporal organization of K-Ras4B, and hence b minus, which is part of the cross- structure resulting in an overall ‘‘LS’’- have also been studied. We showed that PDEd is not able to extract K- shaped topology of individual subunits. The dimer interface protects the hy- Ras4B from model membranes. Instead, an effective delivery of PDEd-solubi- drophobic C termini from the solvent. The characteristic staggering of the lized K-Ras4B to the plasma membrane was proposed. Compared to PDEd, nonplanar subunits results in markedly different fibril ends, termed CaM exhibits additional interaction sites to the G-domain of K-Ras4B and ‘‘groove’’ and ‘‘ridge,’’ leading to different binding pathways on both fibril was shown not to be required for the transport of K-Ras4B to the plasma mem- ends, which has implications for fibril growth. Each monomer that binds to a brane. The influence of CaM on the interaction of GDP- and GTP-loaded K- certain fibril end sees the same interface, in contrast to a true dimeric inter- Ras4B with anionic model raft membranes has been investigated by a combi- face (in the case of a C2 symmetry), where added monomers would alternat- nation of different spectroscopic and imaging techniques. The results suggest ingly see either two identical binding sites or a curb preformed by the that binding of the acidic CaM to the polybasic stretch of K-Ras4B reverses b preceding subunit. The strands are staggered with relation to one another its charge, leading to repulsion of the complex from anionic membranes. in a zipper-like manner. At both fibril ends, the binding site for the addition of subunit i contains contributions of subunits i-1, i-2, i-3, i-4, and i-5, or 937-Symp iþ1, iþ2, iþ3, iþ4, and iþ5, respectively. Therefore, five Ab(1-42) subunits Allosteric Regulation of Small GTPases on Membranes are required to provide the full interface for monomer addition. For a frag- Jacqueline Cherfils. ment of six subunits, the capping subunits would have the same full contact CNRS and ENS Paris-Saclay, Cachan, France. interface as those in an extended fibril. We define this structural element of Lipidated small GTPases and their regulators assemble at the surface of mem- six subunits as the minimal fibril unit. This minimal fibril unit may also be branes to propagate actions in the cell, but an integrated understanding of their the minimal seed size for nucleation as suggested by results from Ab aggre- interactions with the lipid bilayer is still lacking. We addressed this question for gation studies monitored by small angle neutron scattering and analytical ul- Arf GTPases, which orchestrate a variety of regulatory functions in lipid and tracentrifugation. membrane trafficking, and the guanine nucleotide exchange factors (GEF) BPJ 8590_8601 186a Monday, February 19, 2018 Brag2, which controls integrin endocytosis and cell adhesion and is impaired in analyse DNA origami structural offset with angstrom-level precision (2 A˚ ) from cancer and developmental diseases. Brag2 activates Arf GTPases by stimu- single-molecule studies. By combining the approach with multiplexed Exchange- lating the replacement of GDP by GTP, leading to the active, membrane- PAINT imaging, we further demonstrated an optical nano-display with 5Â5nm attached form of Arf which recruits downstream effectors. We showed previ- pixel size and three distinct colours, and

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