Intrinsically Disordered Proteins Drive Membrane Curvature

Sunday, February 28, 2016 domains. Comparison of tau isoforms containing or lacking repeat 2 suggest conformational differences that may relate to differences in affinity and function of these two isoforms. Notably, our results unequivocally support a model of tau with distinct ensemble conformations upon interaction with soluble tubulin and polyanions that promote its aggregation. This work provides insight into conformational changes important to tau loss of function and draws attention to the importance of the role of tau’s conformational plasticity in this process. 1. Elbaum-Garfinkle S and Rhoades E (2012) J Am Chem Soc 134(40):16607– 16613 198-Plat A New Approach to Infer Size and Shape of Disordered Conformations of Proteins from Sm-FRET Data Gregory-Neal Gomes1, Jianhui Song2, Hue-Sun Chan2, Claudiu C. Gradinaru1. 1 Chemical and Physical Sciences, University of Toronto, Mississauga, ON, Canada, 2Molecular Genetics, University of Toronto, Toronto, ON, Canada. The critical biochemical functions of intrinsically disordered proteins (IDPs) are increasingly being recognized but the investigation of biophysical properties of IDPs is only in its infancy. Single-molecule Forster resonance energy transfer (smFRET) is an important tool for studying IDPs. It is commonly utilized to infer structural properties of conformational ensembles by matching experimental average energy transfer efficiency to a value computed from the distribution of end-to-end distances in polymer models. We used polymer theory and computation to address the framework of interpretation of smFRET data for disordered states of proteins. A novel, generally applicable method to infer conformational properties from smFRET histograms was proposed. The new method is grounded in fundamental physical principles and free of the conventional, limiting presumption of a homogeneous conformational ensemble. Extensive sampling of coarse-grained protein chains with excluded volume was performed to generate physically realistic end-to-end distance distributions conditioned on the radius of gyration (descriptor of compactness) and asphericity (descriptor of shape). This sub-ensemblebased approach was used to interpret smFRET data obtained for the yeast the cell-cycle IDP Sic1, and for the DrkN SH3 domain. Encouragingly, the conformational parameters inferred for both proteins, i.e., radius of gyration, hydrodynamic radius and asphericity, are consistent with independent SAXS and NMR measurements. 199-Plat FTIR Study Reveal Intrinsically Disordered Nature of Heat Shock Protein 90 Aihua Xie1, Maurie Balch2, David Neto1, Oliver Causey1, Johnny Hendriks3, Junpeng Deng2, Robert Matts2. 1 Physics, Oklahoma State University, Stillwater, OK, USA, 2Biochemistry & Molecular Biology, Oklahoma State University, Stillwater, OK, USA, 3 Juelich Research Center, Juelich, Germany. Heat shock protein 90 (Hsp90) is a highly conserved chaperone protein that enables the proper folding of a large number of structurally diverse proteins (a.k.a., clients) in the crowded cytosolic environment and plays a key role in regulating the heat shock response. An outstanding question is how Hsp90 accommodates the structural diversity of a large cohort of client proteins? We report ATR FTIR study on structural properties of Hsp90 C-terminal domain (CTD) and their temperature dependences. Our data reveal that within a narrow temperature window, from 35o to 45 oC around physiological temperatures, Hsp90CTD exhibits significant increases in protein aggregation and increases in unordered structures. Binding of Hsp90 inhibitor Clorobiocin elevates the transition temperature of protein aggregation from 38  C (a physiological temperature) to 65  C (far above the physiological temperatures). In striking contrast, the Hsp90 N-terminal domain and middle domain (NTMD) demonstrates no protein aggregation from 20 to 90  C. Despite the intrinsically disordered nature of Hsp90CTD, it retains a protected hydrophobic core at 40  C. We introduce a hydrophobic interlock (HPI) model to account for the functional capability of intrinsically disordered Hsp90. Implications of these results and the HPI model will be discussed in the light of the structural dynamics and client diversity of Hsp90. 200-Plat Physical Principles that Govern the Sequence-Encoded Phase Behavior of Intrinsically Disordered Block-Copolymeric Proteins Alex S. Holehouse, Tyler S. Harmon, Rohit V. Pappu. Biomedical Engineering, Washington University in Saint Louis, St Louis, MO, USA.

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Many intrinsically disordered proteins (IDPs) and sequences with long disordered regions undergo reversible phase transitions such as liquid-liquid demixing or sol-gel transitions to form dense liquids or gel-like phases. Such phases underlie the formation of membraneless compartments within cells. Blockcopolymeric disordered regions appear to be necessary and sufficient to drive phase separation in many systems. The interfacial tension between wellmixed and dense phases can be modulated in different ways: It can be diminished at high salt concentrations, regulated by post-translational modifications such as Ser / Thr phosphorylation, and altered by interactions with RNA molecules. These observations suggest that sequence-encoded electrostatic interactions provide at least part of the driving force for phase separation of IDPs with blocks that are enriched in charged residues. Our goal is to uncover the physical principles that govern the sequence-encoded driving forces for reversible phase transitions of block copolymeric IDPs. Here, we focus specifically on charge-mediated interactions by considering the phase behavior of block-copolymeric polyampholytes and polyelectrolytes. The physics of electrostatically driven phase separation, known as complex coacervation, has been explored in polymer chemistry for synthetic polyelectrolytes. Through a novel combination of lattice-based coarse grain simulations, offlattice coarse-grain simulations driven by machine learning, atomistic simulations, and theoretical insights, we are uncovering a physical framework for sequence-encoded complex coacervation of polyampholytic and polyelectrolytic IDPs. Our focus is on the collective interplay among charge patterning, charge density, chain-length, and the influence of charged versus uncharged residues on the phase behavior, fluidity, and structures adopted by disordered proteins in dense phases. Our findings should enable the systematic design of IDPs with desired phase behavior and proteome-level identification and understanding of how specific types of sequences drive intracellular phase transitions. 201-Plat Cytotoxicity of Prion Protein-Derived Cell Penetrating Peptides is Independent of Amyloid Formation Vineeth Mukundan, Christy Maksoudian, Maria Vogel, Mazin Magzoub. Biology Program, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates. Prion diseases are associated with the conversion of the benign cellular form of the prion protein (PrP(C)) into an abnormally folded and aggregated scrapie isoform (PrP(Sc)), which is also responsible for prion infectivity. We previously showed that peptides derived from the unprocessed N-termini of mouse and bovine prion proteins, mPrP(1-28) and bPrP(1-30), function as cellpenetrating peptides (CPPs) that can efficiently deliver a whole host of cargos. mPrP(1-28) and bPrP(1-30) also exhibited membrane-perturbation effects in model membrane systems. Taken together, the behavior of mPrP(1-28) and bPrP(1-30) provides a potential mechanism for the infectivity and toxicity associated with prion diseases. However, in subsequent studies treatment with mPrP(1-28) or bPrP(1-30) significantly reduced PrPSc levels in prioninfected cells and substantially prolonged the time before infection was manifested after infecting healthy cells with scrapie. To explain these seemingly contradictory results, we correlated the aggregation and toxicity of mPrP(128) and bPrP(1-30) with their cellular uptake and intracellular localization. Although the two peptides have a similar primary sequence, mPrP(1-28) was highly prone to aggregation and formed amyloid fibers, whereas bPrP(1-30) aggregated much less and was non-amyloidogenic. Surprisingly, the nonamyloidogenic bPrP(1-30) induced much higher cytotoxicity than the amyloidogenic mPrP(1-28), suggesting that amyloid formation and toxicity are independent. The toxicity of these peptides was due to membrane perturbation. Interestingly, aggregation and toxicity of the peptides were inhibited by low pH. Following internalization by lipid-raft dependent macropinocytosis, a receptor-independent form of endocytosis, the peptides localized to lysosomes, where aggregation and toxicity were inhibited. Our results shed light on the antiprion mechanism of the peptides and provide a potential site for PrP(Sc) formation. 202-Plat Intrinsically Disordered Proteins Drive Membrane Curvature David J. Busch1, Justin R. Houser1, Carl C. Hayden1, Michael B. Sherman2, Eileen M. Lafer3, Jeanne C. Stachowiak1,4. 1 Biomedical Engineering, University of Texas at Austin, Austin, TX, USA, 2 Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA, 3Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, 4Institute for Cellular and Molecular Biology; University of Texas at Austin, Austin, TX, USA.

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Sunday, February 28, 2016

Many of the key proteins that drive membrane curvature contain large intrinsically disordered protein (IDP) domains of 300-1500 amino acids, including clathrin adaptor proteins and COPII vesicle components. Since IDP domains lack a well-defined secondary structure, these domains have often been regarded as flexible binding domains that do not directly participate in membrane bending. However, we have recently reported the surprising finding that disordered domains are highly potent drivers of membrane curvature (Busch et al., Nat Comms 2015). How can a protein that lacks a defined shape induce membranes to bend? From a biophysical perspective, IDPs, like other polymer chains, occupy a significantly larger volume than globular proteins of equal molecular weight. In line with this thinking, we utilized in vitro measurements of membrane curvature, membrane coverage, and fluorescence correlation spectroscopy to demonstrate that the IDP domains of the endocytic proteins, Epsin1 and AP180, occupy a significantly larger area on membrane surfaces compared to structured domains, making them efficient drivers of membrane curvature through the recently discovered mechanism of protein crowding (Stachowiak et al., NCB 2012, Copic et al., Science 2012). From this perspective, if IDPs drive curvature on the coat side of a membrane, then presenting them on the cargo side should resist internalization through a balance of pressures on the two surfaces of the membrane. Consistent with this model, when we expressed IDPs as cargo molecules on the surface of mammalian cells, steric pressure in the crowded plasma membrane environment excluded them from clathrincoated pits, resulting in their accumulation at the plasma membrane. Building on these findings, our latest work utilizes ligands with bulky disordered polymer chains to sterically retain specific receptors at the plasma membrane, providing a novel physical tool for modulating the dynamics of cellular signaling. 203-Plat Kinetics of Amyloid Fibril Self-Assembly by Direct Observation of Elongation Laurence J. Young, Clemens F. Kaminski. Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom. A hallmark of Alzheimer’s disease (AD) is the deposition of neurotoxic extracellular plaques of amyloid fibrils formed from normally soluble amyloid-b (Ab) peptides. Recent kinetic studies have contributed significantly towards a mechanistic understanding of amyloid fibril self-assembly however it can be challenging to extract microscopic rate constants for the underlying multistep reactions. In this work we develop a new method for imaging Ab42 aggregation reactions in vitro using two-colour fluorescence microscopy. Using this approach we are able to directly measure elongation rate distributions, observe temperature dependent intermittent, or stop-and-go, growth and study the effect of solution conditions on the reaction. By neglecting the primary and secondary nucleation reactions we can unambiguously determine the effect of aggregation inhibitors on the fibril elongation process. 204-Plat From Physiological Fluids to Pathological Gels: Disordered Proteins at the Nexus of Liquid Phase Separation and Neurodegenerative Disease Shana Elbaum-Garfinkle, Nicole Taylor, Clifford P. Brangwynne. Chemical and Bioengineering, Princeton University, Princeton, NJ, USA. Disordered protein domains are emerging as key players in driving the assembly of both pathological fibrous aggregates and functional RNA/protein (RNP) bodies with liquid-like properties. However, the molecular mechanisms governing protein assembly into either liquid-like or solid-like gel states are yet to be understood. Here, we combine soft matter and biochemical approaches to examine the relationship between diverse assembly states of several disordered RNP model proteins. Building on our recent work revealing that protein droplet viscosity and dynamics could be tuned by exogenous substrates or conditions, we investigate the maturation and non-equilibrium dynamics of droplets over time. We use several complementary microrheology approaches, including a new microfluidic platform, to measure precise viscoelastic changes in protein droplet material properties. We also use fluorescence recovery after photobleaching (FRAP) and biochemical aggregation assays to probe the dynamics and solubility states of protein components. Together, this work provides mechanistic insight into the relationship between diverse protein assembly states and the way in which disordered protein and protein/nucleic acid interactions give rise to mesoscopic materials with unique properties.

Platform: Membrane Protein Structure and Folding I 205-Plat ArnT: Structure and Mechanism of the Aminoarabinose Transferase Responsible for Resistance to Polymyxin-Class Antibiotics Vasileios I. Petrou1, Carmen M. Herrera2, Kathryn M. Schultz3, Oliver B. Clarke4, Jeremie Vendome4,5, David Tomasek1, Surajit Banerjee6, Kanagalaghatta R. Rajashankar6, Brian Kloss7, Edda Kloppmann8, Burkhard Rost8,9, Candice S. Klug3, M. Stephen Trent2, Lawrence Shapiro4, Filippo Mancia1. 1 Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA, 2Department of Infectious Diseases, University of Georgia, College of Veterinary Medicine, Athens, GA, USA, 3Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA, 4 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA, 5Department of Systems Biology, Columbia University, New York, NY, USA, 6Department of Chemistry and Chemical Biology, Cornell University, NE-CAT, Advanced Photon Source, Argonne, IL, USA, 7New York Consortium on Membrane Protein Structure (NYCOMPS), New York Structural Biology Center (NYSBC), New York, NY, USA, 8Department of Informatics, Bioinformatics and Computational Biology, Technische Universita¨t Mu¨nchen (TUM), Garching, Germany, 9 Institute for Advanced Study (TUM-IAS), Technische Universita¨t Mu¨nchen (TUM), Garching, Germany. Polymyxins are cationic peptide antibiotics, once discarded due to toxicity concerns, that have been revived in the clinic and are widely used to treat multidrug resistant (MDR) infections. Resistance to polymyxin-class antibiotics is a growing concern, as a result, one that can critically impair our ability to combat MDR infections. ArnT (4-amino-4-deoxy-L-arabinose transferase) is an integral lipid-to-lipid glycosyltransferase that modifies lipid A, the lipid component of bacterial lipopolysaccharide (LPS). ArnT is located in the inner membrane of Gram-negative bacteria and catalyzes the transfer of a modified arabinose moiety from undecaprenyl phosphate to lipid A. The addition of the arabinose moiety causes a charge modification of the bacterial outer membrane, limiting its interactions with cationic peptides, and enabling bacteria to develop resistance to polymyxin-class antibiotics and natural antimicrobial peptides. Here we report crystal structures of ArnT from a gram-negative bacterium, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 ˚ resolution, respectively. ArnT consists of a transmembrane domain and 3.2A with thirteen transmembrane helices and a periplasmic soluble domain. The overall fold is reminiscent of protein glycosyltransferases from bacteria and archaea, but ArnT possesses unique features that are related to its function as a lipid glycosyltransferase. Notably, we identify cavities for both lipidic substrates, accessible to the membrane environment, that converge at the active site. We observe a significant coil-to-helix structural transition upon binding of undecaprenyl phosphate that stabilizes the carrier lipid near the active site and expands the lipid A cavity, likely enabling subsequent binding of lipid A. Finally, we utilize a polymyxin resistance assay to investigate the role of particular residues in light of the structures, and propose a model for catalysis by ArnT family enzymes. 206-Plat Structural Studies of the Human Kappa Opioid Receptor Active State Conformations Ming-Yue Lee1, Nilkanth Patel2, Vsevolod Katritch2, Raymond C. Stevens1, Vadim Cherezov1. 1 Chemistry, University of Southern California, Los Angeles, CA, USA, 2 Molecular and Computational Biology, University of Southern California, Los Angeles, CA, USA. The crystal structure of the human kappa opioid receptor (hKOR) in complex ˚ (Wu et al, 2012, Nawith antagonist JDTic had been previously solved to 2.9 A ture 485, 327-332). From this crystal structure, key receptor-ligand interactions were observed and extended via modeling to GNTI, b-NNTA, and other hKOR ligands. However, a major hurdle still remains in elucidating hKOR in different states of activation. Having this information would lead to a more comprehensive understanding of hKOR activation and function, informing design of more efficient analgesics with reduced side effects. Specifically, we seek to utilize selective hKOR ligands to trap hKOR in conformational states that correlate to the properties of each ligand. Here we present our work in progress on the