A Dead-Box Protein Acts through RNA to Promote HIV-1 Rev-RRE Assembly

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368-Pos Board B133 CRISPR-Cas9: Computational Insights Toward Improved Genome Editing Giulia Palermo1, Yinglong Miao1, Ross C. Walker1, Martin Jinek2, J. Andrew McCammon1. 1 Department of Pharmacology, University of California at San Diego/ Howard Huges Medical Institute, La Jolla, CA, USA, 2Department of Biochemistry, University of Zurich, Zurich, Switzerland. Life sciences are undergoing a transformative phase due to an emerging genome-editing technology based on the RNA-programmable CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system. In this system, the endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. This missing aspect hampers the precise structure-based design of CRISPR-Cas9 genome-editing tools with improved specificity. Here, we report the first biophysical study – based on extensive multi-microseconds molecular simulations integrated with structural data – revealing the conformational plasticity of Cas9 and identifying the key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We identify a remarkable conformational plasticity as an intrinsic property of the nuclease HNH domain, being a necessary factor allowing for the HNH domain repositioning during catalysis. More importantly, we disclose a key role of the non-target DNA during the process of activation of the HNH domain, showing how the non-target DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Our outcomes further suggest new and precise proteinengineering modifications, which are of fundamental importance for the rational design of more effective genome-editing tools. Overall, these novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features and at the improvement of biological applications of the CRISPR-Cas9 system. 369-Pos Board B134 Structural Insights into G-tract Recognition by the hnRNP H-RNA Recognition Motif Srinivasa R. Penumutchu. Chemistry, Case Western reserve University, Cleveland, OH, USA. The heterogeneous nuclear ribonucleoprotein H (hnRNP H) family of proteins are involved in RNA splicing of cellular and viral mRNAs. These proteins function as both splicing activators and repressors. The hnRNP H family proteins have previously been found to interact with poly-G sequences (G-tracts) of cellular and viral mRNAs using quasi RNA recognition motifs (qRRMs) including human immunodeficiency virus (HIV). hnRNP H proteins are composed of three qRRMS, which are separated by linkers and two Glycine rich domains at C-terminal. The qRRM1 and qRRM2 domains are located at the N-terminus and separated by 10-residue linker, whereas qRRM3 is located towards the C-terminus. To gain structural insights into hnRNP H protein, here we solved the solution structure of the HRRM12 domain of hnRNP H using NMR spectroscopy. We used paramagnetic relaxation enhancement (PRE) and Residual dipolar couplings (RDC) to obtain distance restraints and orientational restraints between the HRRM1 and HRRM2 domains. T1, T2 and NOE experimental data is consistent with calculated structure of HRRM12 domain and reveals that HRRM12 adopt the compact (closed) structure. To better understand principals of hnRNP H-RNA recognition, we systematically screened G-tracts of RNA oligos against HRRM12 by using isothermal titration calorimetry (ITC) and NMR spectroscopy. We examined the minimal G-tract RNA (–GGG–) sequence requirements for HRRM12 binding. The HRRM12 domain of hnRNP H exhibited no substantial differences in binding affinities for G-tracts of RNA (AGGGX) and HRRM12 residues involved in binding G-tracts of RNA were detected by NMR chemical shift perturbation experiments and a data-driven model of the complex was determined using HADDOCK. Taken together, this study provides the molecular insights for better understanding the role of the qRRM domains of hnRNP H in RNA splicing of cellular and viral mRNAs. 370-Pos Board B135 Blind Predictions of RNA/Protein Relative Binding Affinities Kalli Kappel, Inga Jarmoskaite, Pavan P. Vaidyanathan, William J. Greenleaf, Daniel Herschlag, Rhiju Das. Stanford University, Stanford, CA, USA. Interactions between RNA and proteins are pervasive in biology, shaping processes such as mRNA translation, localization, and alternative splicing. Developing a predictive understanding of the energetics of these systems would allow

us to model biologically relevant mutations of these interactions and ultimately design novel interactions. Despite recent advances in high throughput experimental technologies that measure the energetics of these systems, quantitative computational prediction of relative RNA/protein binding affinities has remained a challenge. This is partly due to the observation that computational binding affinity prediction methods typically break down when the molecules are highly flexible or undergo significant conformational changes, situations that often arise in RNA/protein binding. Here, we present a novel framework within Rosetta for predicting RNA/protein relative binding affinities that begins to address this issue. Specifically, we show that the nearest neighbor energies, which are typically used for RNA secondary structure prediction, can be used to approximate the unbound free energy of the RNA, thus eliminating the need to explicitly account for the flexibility of the unbound RNA or conformational changes of the RNA upon binding. Using this method of calculating the unbound RNA free energy significantly improves the prediction accuracy over a more typical 3D structure-based approach. We optimized this method using a subset of published MS2 coat protein affinities and ultimately made predictions for the system with 1.11-1.28 kcal/mol root mean square (RMS) error. Additionally, we show that this method is able to predict relative binding affinities for four diverse RNA/protein systems with 1.48 kcal/mol RMS error. Finally, to more rigorously assess this method, we independently measured and made blind predictions for PUF3 and PUM2 binding affinities with RMS errors of 1-2 kcal/mol, which is comparable to the accuracy achieved by prediction methods for other types of systems. 371-Pos Board B136 Biophysical Studies of Liposome Encapsulated Pokeweed Antiviral Protein and its use as a HIV Therapeutic O’Jay Stewart1, Artem Domashevskiy2. 1 CUNY - John Jay College, Manhattan, NY, USA, 2Department of Sciences, CUNY - John Jay College, Manhattan, NY, USA. Human Immunodeficiency Virus (HIV) is a virus that attacks the human immune system, compromising its effect in regards to disease prevention. HIV results in the destruction of CD4 cells, which are vital in the defense against human immune responses. HIV can severely damage the immune system and lead to Acquired Immunodeficiency Syndrome (AIDS). There is presently no cure or effective HIV vaccine and as a result it is important to seek alternative measures in the defense against HIV/AIDS. Pokeweed Antiviral Protein (PAP), a protein isolated from pokeweed plant, Phytolacca americana, provides a new and profoundly promising direction in the field of HIV/AIDS research. PAP plays a vital role in the immune system of pokeweed and is a ribosome inactivating protein (RIP), inhibiting viral protein production. PAP possesses antiviral properties, and reduces the infectivity of many plant and animal viruses, including HIV-1. My project focuses on mechanisms using liposomal encapsulated PAP, targeted to infected cells as a therapeutic for HIV infection. Steady state fluorescence was used to identify the thermodynamic parameters of interaction between PAP and HIV RNA. PAP binds to the m7 GTP cap and the 50 UTR region of HIV RNA. Various isoforms of PAP were tested and the optimal affinity was identified as a Km of 20.09 nM. Fluorescence observed V.S [m7 GTP] was plotted through nonlinear regression using the Prism 6.0 program. Varying liposomes were prepared in order to identify the optimal characteristics for PAP encapsulation. Liposomes were lyophilized through rotary evaporation and later extruded to ensure homogeneity. Fluorescent titration enables the testing of liposomal PAP encapsulation efficiency. It was concluded that a 1/1 v% mixture of liposomes DOPE-DOTAP is most efficient for PAP encapsulation. These accomplishments will enable further modification of the liposomes by covalently conjugating HIV-specific monoclonal antibodies (anti-pg120 and/or anti-pg41), thus facilitating the selectivity of the liposomal PAP to the target tissues (HIV-infected CD4þ lymphocytes). With the aforementioned completed the basis for PAP-liposome interactions can be established and also the modulation of these interactions in order to target HIV-infected cells. 372-Pos Board B137 A Dead-Box Protein Acts through RNA to Promote HIV-1 Rev-RRE Assembly Rajan Lamichhane, John A. Hammond, Raymond F. Pauszek, Ingemar Pedron, Edwin van der Schans, James R. Williamson, David P. Millar. Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. The HIV-1 Rev (Regulator of Expression of Virion) protein activates nuclear export of unspliced and partially spliced viral mRNAs, which encode the viral genome and the genes encoding viral structural proteins. Rev interacts with a highly conserved region, the Rev Response Element (RRE), located within

Sunday, February 12, 2017 the viral mRNA. In order to activate nuclear export, multiple Rev proteins must assemble on the RRE. The host DEAD box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. We used a single-molecule assembly assay utilizing immobilized full length RRE and fluorophore-labeled Rev to monitor each step of Rev-RRE assembly, in the presence or absence of DDX1. More Rev monomers were observed to bind to the immobilized RRE in the presence of DDX1, indicating that DDX1 promotes oligomeric Rev-RRE assembly. Further experiments using specific DDX1 mutants that are defective in either Rev binding or RNA binding indicate that DDX1 must be capable of associating with RNA in order to promote assembly of the Rev-RRE complex. Single-molecule Fo¨rster resonance energy transfer (smFRET) experiments show that DDX1 transiently interacts with the RRE and that both DDX1 and Rev can occupy the same RRE molecule. Taken together, these results suggest that DDX1 acts as an RNA chaperone, folding the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process. Supported by NIH grant GM082545. 373-Pos Board B138 Deciphering the Action Mechanism of DDX3: An RNA Helicase Implicated in Cancer Propagation and Pathogenic Viral Infection Anthony F. Moore, Aliana Lopez, de Victoria, Eda Koculi. Chemistry, University of Central Florida, Orlando, FL, USA. DDX3 is a human DEAD-box RNA helicase implicated in crucial cellular processes including translation initiation, ribosome assembly, RNA transport, and microRNA processing. Consequently, DDX3 is implicated in many viral infections and cancer cell metabolism. Our goal is to obtain a detailed understanding of DDX3’s mechanism of action and employ this understanding to discover DDX3 inhibitors that would serve as lead compounds for drugs that halt viral infections and cancer cell metabolism. While DDX3 is required for many viral infections and cancer cell propagations, it is not essential for healthy cell metabolism, making DDX3 an ideal anticancer and antiviral drug target. Like all the members of the DEAD-box family of enzymes, DDX3 uses ATP binding and hydrolysis to unwind short double-stranded RNA helices. Our data show that different from many members of DEADbox family of enzymes, monomeric DDX3 is unable to perform RNA unwinding, and a multimeric DDX3 complex is required to support DDX3’s helicase activity. Furthermore, our data suggests that the single-stranded-doublestranded RNA junction promotes the formation of the DDX3 multimer. We are in the process of performing mutagenesis studies combined with crosslinking and mass spectrometry to determine the DDX3 amino acids implicated in mulitmer formation and the amino acids that come in direct contact with RNA during the DDX3 catalytic cycle. These experiments would produce information both on DDX3’s mechanism of action, and elucidate why some DEAD-box proteins have evolved to act as mulitmers. Lastly, we have found four natural compounds that are specific inhibitors of DDX3 ATPase activity. These compounds will be used as probes to decipher DDX3’s action mechanism and could have translational potential as drugs that stop various viral infections and cancer progression. 374-Pos Board B139 Interaction of PKR with Single Stranded RNA Christopher B. Mayo, James L. Cole. Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA. Although the antiviral kinase PKR was originally believed to interact with only duplex RNAs, evidence has accumulated that the enzyme can be activated by a variety of structured RNAs. A potent PKR activating motif consists of a short stem loop containing single stranded tails (ss-dsRNA). The ssRNA tails contribute to binding and activation. However, PKR does not contain a canonical ssRNA binding domain. Here, we demonstrate that isolated ssRNAs interact with PKR. Both homopolymeric (rU)30 and heteropolymeric 30 nt ssRNAs bind with micromolar dissociation constants. Addition of a 50 -triphosphate slightly enhances binding affinity. A homopolymeric (dT)30 ssDNA binds more weakly than (rU)30, indicating a contribution of the 20 OH moiety. PKR contains a conserved region N-terminal to the kinase that is enriched in basic residues. ssRNA binds to a construct containing the basic region and kinase and also binds to the isolated dsRNA binding domain. Both full length PKR and the basic region/kinase domain construct are weakly activated by ssRNA. However, the isolated kinase is not activated and does not bind ssRNA. Photocrosslinking measurements were performed using a ss-dsRNA containing 4-thiouridine and PKR constructs with a TEV protease cleavage site at different positions between the kinase and dsRNA binding domain. Analysis of the products following crosslinking and TEV cleavage demonstrates that that the basic region interacts with ss-dsRNA in the context of full length PKR. Our results support a model where PKR activation by

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Membrane Dynamics I 375-Pos Board B140 Surfactant Micelle Self-Assembly with Coarse-Grained Martini Standard Water and Polarizable Water Eric Sefah, Blake Mertz. Chemistry, West Virginia University, Morgantown, WV, USA. Molecular dynamics (MD) simulations of proteomicelle complexes are useful for the investigation of membrane protein dynamics and function. However, modeling assembly and dynamics of complex oligomeric systems require length- and timescales that are traditionally inaccessible to all atom MD simulations. Coarse grained (CG) force fields such as MARTINI decrease spatial resolution of a system, making biologically relevant time scales for phenomena such as membrane protein oligomeric assembly accessible [1]. In order to apply CG force fields to the study of large proteomicelle complexes it is important to test their ability to reproduce experimentally observed properties. In this study we have characterized the effects of the nonpolarizable and polarizable water models of the MARTINI force field [2] on assembly of surfactant micelles. The two surfactant systems studied were a zwitterionic detergent (n-dodecylphosphocholine (DPC)) and nonionic detergent (n-dodecyl-b-D-maltoside (DDM)). From a system of 50, 100, 150 and 200 random detergent molecules, stable micelles formed with variable sizes for both models, in general agreement with experimental aggregation numbers. However, the polarizable water model formed larger micelles than the normal water model in a few cases. In addition, the polarizable water model formed stable compact micelles in shorter times. [1] Marrink SJ, et al. J. Phys. Chem. B. 2007;111:7812. [2] Yesylevskyy SO, et al.PLoS Comp. Biol, 2010;6:e1000810. 376-Pos Board B141 Simulations of Glycerol and its Effect on the Phase and Behaviour of DPPC Monolayers Jemma L. Trick1, Wachirun Terakosolphan2, Ben Forbes2, Christian D. Lorenz1. 1 Physics Department, Kings College London, London, United Kingdom, 2 Institute of Pharmaceutical Science, Kings College London, London, United Kingdom. Lung Surfactant (LS), a monolayer coating the alveolar surface undergoes changes during breathing. Glycerol, a known cryo-protectant is known to induce folding in LS, stiffening in model LS monolayers, modulate area per lipid, and the transition temperature of such systems experimentally. Using atomistic molecular dynamics (MD) simulations, we model LS as a pure dipalmitoylphosphatidylcholine (DPPC) monolayer, in which the concentration of glycerol and simulation temperature is varied to investigate the molecular basis of such variations in behaviour. Our Simulations suggest a possible dehydration stage of DPPC head-groups under high concentrations of glycerol, which result in a change in DPPC transition temperature (6). This change could influence the use of glycerol in possible aerosol devices and the permeability of the modulated monolayer. 377-Pos Board B142 Protocol and Validation of CHARMM-GUI Hex Phase Builder Andrew H. Beaven1, Alexander J. Sodt2, Richard W. Pastor3, Wonpil Im4. 1 Chemistry, The University of Kansas, Lawrence, KS, USA, 2Unit on Membrane Chemical Physics, National Institutes of Health, Bethesda, MD, USA, 3Laboratory of Computational Biology, National Institutes of Health, Bethesda, MD, USA, 4Department of Biological Sciences and Bioengineering Program, Lehigh University, Bethlehem, PA, USA. It is becoming increasingly apparent that protein dynamics, conformation, and therefore, function are dependent on the stresses within the bilayer – one of which being bending frustration. The amount of bending frustration is dependent on the bending modulus (kc) and spontaneous radius of curvature (R0–1) for a given lipid composition. These quantities are difficult to obtain computationally, particularly by all-atom molecular dynamics (MD) simulation. For many years, experimentalists (and much more recently, simulators) have used the lipid hexagonal phase to obtain these quantities. The inverse lipid hexagonal phase exists in high temperature and low water domains of the certain lipid’s phase diagram. In this phase, lipids aggregate with their hydrophilic heads oriented along hexagonally packed water pores. Although this phase spontaneously assembles, the equilibration time necessary for this type of process is unfeasible for typical all-atom MD simulations. Here, a simple and reproducible methodology, Hex Phase Builder, is described and validated as