The Allosteric Site is Required for Voltage Dependence of Muscarinic GPCRs

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flies to human that it is implicated in multiple roles, including division of chromatin, gene activation, gene repression, and genome looping among others. CTCF contains 11 zinc fingers that can bind up to 35 bp of DNA including the well-conserved CCCTC sequence. Moreover, the binding sites orientation may play an important role in the regulation of enhancer-promoter choice, and the direction of looping formation. Here, we have used single molecule fluorescence resonance energy transfer (smFRET) to monitor and characterize CTCF-mediated DNA lopping. To this aim, we have designed a DNA substrate with a fluorophore-labelled (Cy3 and Cy5) binding site located at each end, such that loop formation brings together the two ends of the molecule thereby we will be able to observe FRET signal. Our data show that loop formation is only observed in the presence of two sequence specific binding sites and CTCF. Loop formation probability increases with protein concentration, yielding a dissociation constant of 10 nM and a Hill coefficient 1.6 in agreement with looping induced by dimers. Loop formation probability depends strongly on the distance between the binding sites, similar to previously reported models for the bacterial Lac operator. Contrary to what is observed in cells, our data show that CTCF does not favor any particular binding site orientation in vitro. Our results indicate that the preferred looping orientation observed in vivo likely results from additional regulatory looping factors. 837-Plat Protein-Mediated Loops in Supercoiled DNA Create Large Topological Domains Yan Yan1, David D. Dunlap1, Fenfei Leng2, Laura Finzi1. 1 Department of Physics, Emory University, Atlanta, GA, USA, 2Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA. Topological domains are structural units of practically all DNA genomes. They are formed of segments with constrained ends, usually by proteins. If these boundary elements restrict the free rotation of the DNA, they may be torsionally isolated. The existence of the topological domains helps to compact the DNA, reduce the amount of DNA that is relaxed by nicking, specifically regulate cellular processes, and partition the genome into active and non-active regions. A naı¨ve expectation is that the size of such domains would be as large as the distance between the protein binding sites. This appears to be the case in relaxed DNA. To investigate this magnetic tweezers were used to arbitrarily supercoil DNA segments containing high affinity LacI binding sites separated by 400 bp. As shown previously for lambda repressor-mediated loops, slight tension abolished looping unless the DNA was supercoiled and additional supercoiling compensated for increasing tension up to a threshold. Furthermore, the LacI protein junction of the loop was an effective barrier to the diffusion of supercoils even in DNA subject to the maximum torsional strain that can be produced under 0.5 pN of tension. Finally, loops that formed in supercoiled DNA often created topological domains that extended past the loop segment boundaries. Thus in considering loop-stabilized, topological domains it is important to consider the possibility that they include and affect the availability of sites outside the loop in supercoiled DNA. 838-Plat Toward Direct Observation of the DNA Binding Dynamics of Monomeric Type IIP Restriction Endonucleases Candice M. Etson. Physics Department, Wesleyan University, Middletown, CT, USA. Restriction endonucleases (REases) are enzymes that cleave duplex DNA at a recognition site specified by a particular nucleotide sequence. These enzymes are found in a wide range of bacterial species, and serve as a defense against infection by phage. Type IIP REases are typically antiparallel homodimers that recognize a palindromic DNA sequence and cleave within or close to the recognition site. However, some Type IIP REases cleave pseudopalindromic sequences, and some of these may be active as monomers. It is hypothesized that these monomeric REases sequentially cleave the two complementary strands of duplex DNA during a single DNA binding event. Since the two strands of DNA are antiparallel, after cleaving one strand, a monomeric REase would be required to rotate about the axis perpendicular to the DNA to be properly oriented to cleave the second strand. This type of reorientation is known a ‘‘flipping’’. Although only a small number of proteins have been observed reorienting in this way, protein flipping may play an important role in binding to a specific DNA sequence. Direct observation of flipping during DNA cleavage will greatly improve our understanding of how monomeric REases mediate double strand breaks. We are currently using total internal reflection fluorescence (TIRF) microscopy to observe REase-mediated cleavage of quantum dot-labeled DNA at the single-molecule level. In our highly multiplexed assay, the disappearance of a quantum dot indicates cleavage of the DNA that tethers it to a functionalized glass coverslip. We plan to utilize single-molecule Fo¨rster resonance energy

transfer (smFRET) to trace individual monomeric REases in real time through the entire process of DNA cleavage from substrate binding to product release. Our observations should provide significant insight into the mechanism by which these enzymes cleave duplex DNA. 839-Plat Single Molecule Studies on G-Quadruplex, Protein, and Small Molecule Interactions Hamza Balci, Sujay Ray, Jagat Budhathoki, Parastoo Maleki. Physics, Kent State University, Kent, OH, USA. Recent single molecule studies have revealed a number of significant insights on the mechanism of protein and G-quadruplex (GQ) interactions. In particular, our work on GQ interactions with single stranded DNA binding proteins, such as RPA and POT1, and helicases, such as BLM and RECQL5, showed significant variations in terms of the underlying dynamics and efficacy of these molecules in unfolding GQ structures. On the other hand, GQ-stabilizing small molecules have attracted attention due to their potential use as telomerase inhibitors and anticancer drugs. Such small molecules are expected to promote GQ folding and stability while inhibiting the activity of proteins that unfold GQ. A comparative single molecule study of how such small molecules influence GQ folding and stability and inhibit the activity of GQ-destabilizing proteins will be presented. 840-Plat High-Resolution Single Molecule Rotation Tracking of RecBCD using DNA Origami Rotors Benjamin D. Altheimer1, Pallav Kosuri2, Mingjie Dai1, Peng Yin3,4, Xiaowei Zhuang2,5. 1 Biophysics, Harvard University, Boston, MA, USA, 2Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA, 3Wyss Institute, Harvard University, Boston, MA, USA, 4Systems Biology, Harvard University, Boston, MA, USA, 5Physics, Harvard University, Cambridge, MA, USA. We have invented a new high throughput, high resolution single molecule method that enables tracking the rotation of DNA molecules with millisecond time resolution. Origami Rotational Beacon Image Tracking (ORBIT) uses DNA origami rotors that can be ligated to a DNA duplex of interest. We track the rotation of the DNA duplex, as amplified by the DNA origami, using fluorescent labels on one arm of the origami. ORBIT does not rely on an externally applied force but instead achieves high spatiotemporal resolution mainly due to the low drag of the origami rotor and the stiffness of the short attached DNA duplex. Using ORBIT, we report the first direct measurements of DNA rotation by the helicase RecBCD. We observe pausing and backtracking and account for our observations with a simple translocation model. We also report high resolution measurements of RecBCD initiation, discovering unexpected reversibility in DNA unwinding and rewinding prior to translocation. Our results using substrates with single stranded overhangs indicate that the RecB motor domain is responsible for initiating processive translocation.

Platform: Membrane Receptors and Signal Transduction II 841-Plat The Allosteric Site is Required for Voltage Dependence of Muscarinic GPCRs Anika Hoppe1, Moritz B€unemann2, Andreas Rinne1. 1 Department of Cardiovascular Physiology, Ruhr-University Bochum, Bochum, Germany, 2Department of Pharmacology and Clinical Pharmacy, Philipps-University Marburg, Marburg, Germany. G-protein coupled receptors (GPCRs) encompass the largest group of membrane proteins in eukaryotes and regulate numerous intracellular pathways. Class A GPCRs are activated by binding of specific agonists to an orthosteric binding site, which is formed by the seven trans-membrane helices. In contrast, so called allosteric modulators bind to a site on the extracellular surface, the allosteric site. There is an interaction of both sites to modify receptor function and allosteric modulators either enhance or attenuate receptor signaling. Previous work from our laboratory showed that muscarinic receptors (M-Rs) are voltage sensitive. Voltage dependence was only evident for active receptors and may represent allosteric modulation of M-Rs. In this study we compared the voltage dependencies of M1-Rs and M3-Rs to allosteric modulation of both receptors. Changes in receptor signaling were quantified in single HEK 293 cells with a FRET biosensor for the Gq protein cycle. In the presence of acetylcholine (ACh), a depolarization of the membrane from
90 mV to þ40 mV potentiated M1-R signaling, whereas it attenuated M3-R signaling. Likewise, the positive allosteric modulator BQCA potentiated ACh/M1-R signaling, whereas the negative allosteric modulator Gallamine attenuated

Monday, February 13, 2017 ACh/M3-R signaling. In terms of kinetics and amplitude, receptor modulation by voltage or by allosteric compounds was surprisingly similar. Because the orthosteric site is well conserved, but the allosteric site varies among M-R subtypes, we constructed ‘‘allosteric’’ M1/M3-R chimeras and subjected them to membrane depolarization. Some chimeras were almost insensitive to voltage, indicating that extracellular receptor structures affect the mechanism of voltage dependence. Moreover, a single point mutation that disrupts the cooperativity between the orthosteric and allosteric site removed the voltage sensitivity of M3-R. We conclude that the allosteric site is involved in a mechanism that connects voltage sensitivity and muscarinic receptor function. 842-Plat Probe Activation Mechanism of 6TM Variants of Mu-Opioid Receptor by a Morphine Derivative (IBNtxA) using All-Atom Molecular Dynamics Simulation with Explicit Membrane Safaa Sader, Anant Kumar, Chun Wu. Chem & Biochem, Rowan University, Glassboro, NJ, USA. Morphine, activating Mu Opioid receptors (MOR-1), produces powerful and immediate analgesic effects. However, morphine use is limited by its high addiction tendency and other serious adverse effects. Recent studies have shown that IBNtxA, a morphine derivative, is 10-fold more potent and has a better safety profile than morphine. The animal studies indicated that the IBNtxA analgesics was from the activation of the truncated spice 6TM variants of MOR-1 in which TM1 is removed. Interestingly, IBNtxA was not able to activate the full length 7TM variants of MOR-1 and morphine was only able to activate 7TM variants but not 6TM variants. There is no high resolution structure of 6TM variants, and the activation mechanism of 6TM variants by IBNtxA remains to be elusive. In this study we used homology modeling, docking and molecular dynamics (MD) simulations to study a representative 6TM variant (G1) and the full length 7TM of human MOR-1 in complex with IBNtxA and morphine. The structure model of G1 was obtained by homology modeling based on X-ray solved crystal structure of active mouse MOR-1 bound to the agonist BU72 (PDB id: 5C1M). Our ms MD data shown that either TM1 truncation (from 7TM to 6TM) or ligand modification (from morphine to IBNtxA) alone caused the loss of key morphine-7TM interactions that are required for the receptor activation and the receptor conformation located at TM2, TM3, TM6 and TM7. However, when both perturbations occur in 6TM-IBNtxA complex, the key interactions and receptor active conformation were maintained. Our energetic, structural and dynamic data consistently supports our explanation. 843-Plat Decay of an Active GPCR: Conformational Dynamics Govern Agonist Rebinding and Persistence of an Active, Yet Empty, Rhodopsin State Christopher T. Schafer1, Jonathan F. Fay1, Jay M. Janz1,2, David L. Farrens1. 1 Biochemistry, Oregon Health and Science University, Portland, OR, USA, 2 Pfizer Rare Disease Research Unit, Cambridge, MA, USA. GPCRs are a major pharmaceutical drug target. One exception has been the visual photoreceptor rhodopsin, often considered ‘‘different’’ because its light-sensitive ligand, retinal, is covalently attached to the receptor. Recently however, in contrast to prior assumptions, we discovered rhodopsin can behave like a ‘‘classical’’, diffusible ligand binding GPCR, with the receptor conformation preferentially governing the binding of the agonist (all-trans retinal, ATR) and the antagonist (11-cis retinal, 11CR) (1). Building on that work, we recently gained two new insights into the role receptor conformational dynamics play during release of the agonist (ATR) from rhodopsin (2). First, we show that ATR release from light-activated rhodopsin is not irreversible, but rather, ATR can continually release and rebind to any receptor remaining in an active-like conformation. Second, we find that during decay of the photo-activated state, an active-like, yet empty, receptor conformation can transiently persist even after retinal release, before the receptor ultimately collapses into an inactive conformation. This conclusion is based on studies using a novel, time-resolved fluorescence approach (TrIQ) that show a small, but reproducible, lag between ATR leaving the protein and the return of a key part of the receptor (transmembrane helix 6, or TM6) to an inactive conformation. Accelerating ATR dissociation through either chemical or mutagenesis methods further increased this time-lag. These new findings: i) show the agonist ATR can bind to rhodopsin in equilibrium like a traditional GPCR, ii) indicate that an active GPCR conformation can persist even after agonist release, and iii) raise the possibility that under some conditions the antagonist 11CR may exhibit similar equilibrium binding to rhodopsin, and iv) show rhodopsin could potentially be targeted by traditional pharmaceutical-based treatments using drugs that can outcompete retinal equilibrium binding. I will briefly discuss these findings, and talk about their potential implications regarding a ‘‘universal binding site’’ we recently identified on rhodopsin that is used to bind G-proteins, arrestins and kinases (3).

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References: 1. Schafer CT, Farrens DL (2015) J Biol Chem. PMID: 25451936; 2. Schafer, C., Fay, JF, Janz, JM, Farrens, DL (2016) Proc Natl Acad Sci U S A. PMID: 27702898; 3. Jones Brunette AM, Sinha A, David L, Farrens DL (2016) Biochemistry. PMID:27078130. 844-Plat Analysis of Receptor Tyrosine Kinase and G-Protein Coupled Receptor Signaling Dynamics on Micro-Structured Surfaces Peter Lanzerstorfer1, Yosuke Yoneyama2, Fumihiko Hakuno3, Diana Zindel4, Ulrike M€uller1, Cornelius Krasel4, Moritz B€unemann4, Otmar Ho¨glinger1, Shin-Ichiro Takahashi3, Julian Weghuber1. 1 University of Applied Sciences Upper Austria, Wels, Austria, 2Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan, 3Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan, 4Philipps-Universit€at Marburg, Marburg, Germany. Membrane-localized proteins are essential to transmit signals into the cell. An important issue is the interaction of these proteins with cytosolic proteins. To quantify such often short-lived interactions we have introduced a method based on the combination of micro-structured surfaces and TIRF microscopy.(1,2) We have used the assay to validate the efficacy of medically relevant receptor tyrosine kinase (RTK) modulators.(3) Bait epidermal growth factor (EGF) receptor molecules were forced into microscopic domains, while monitoring corecruitment of fluorescent intracellular prey Grb2 molecules. Pretreatment with pharmacologically active ingredients used for the treatment of human cancers significantly reduced this interaction. A similar approach was used for the quantitative analysis of the interaction between different insulin receptor substrate (IRS) proteins and the insulin/IGF-I receptor.(4) The micro-patterning technique enabled the measurement of equilibrium associations and interaction dynamics of these molecules with high specificity. We revealed that several domains of IRS critically determine the turnover rate of the receptors. Furthermore, we found significant differences among IRS proteins in the strength and kinetic stability of the interaction with the receptors, which could account for the diverse IRS functions. Finally, we studied the interaction of the GPCR ß2-adrenoceptor (ß2AR) with arrestin-3.(5) By measuring arrestin-3 recruitment and the stability of arrestin-3-receptor complexes in real time using FRAP analysis on micro-patterned surfaces, we could demonstrate that arrestin-3 dissociates quickly and almost completely from the b2AR. Recently, we have implemented micro-structured and functionalized multi-well plates. This development step sets a milestone in terms of throughput rates of our methodology. References: 1. Schwarzenbacher, M. et al., 2008. Nature Methods. 2. Sevcsik, E. et al., 2015. Nature communications. 3. Lanzerstorfer, P. et al., 2014. PLOS One. 4. Lanzerstorfer, P. et al., 2015. FEBS Journal. 5. Zindel, D. et al., 2015. Molecular Pharmacology. 845-Plat Class I Cytokine Receptors: Towards the Inside Helena Steinocher1, Katrine Bugge1, Louise Fletcher Nikolajsen1,2, Kresten Lindorff-Larsen1, Andrew Brooks3, Birthe Brandt Kragelund1. 1 Department of Biology, University of Copenhagen, Copenhagen, Denmark, 2 Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark, 3Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia. The Growth Hormone Receptor (GHR) is a member of the Class I Cytokine Receptor family and comprises three different domains, an extracellular domain (ECD), a cell membrane spanning helix constituting the transmembrane domain (TMD) and an intracellular domain (ICD). Growth Hormone (GH) binds extracellularly to two receptors thereby activating two Janus Kinases 2 (JAK2) bound to the ICDs. Despite these receptors being studied for decades it remains an enigma how the signal of the extracellular binding event is translated into the cell. It has been suggested that the TMDs rearrange upon receptor activation, but the TMD dimer conformation of the inactive or active states is not known. We have investigated the structure and membrane interaction of the GHR TMD and ICD by NMR exploiting our new method for generation of isotopically labelled TMD. Reconstitution in DHPC detergent micelles allowed for assignments of the NMR backbone chemical shifts of the TMD and chemical shift analysis of the isolated GHR ICD indicates a fully intrinsically disordered ICD, including the 9 residues separating the TMD and the JAK2 binding site. Thus the question arises how the mechanical force of a hormone induced ECD and TMD rearrangement is translated to the JAK2 binding site, leading to its activation. Lipid binding sites in the GHR ICD juxtamembrane region have been proposed which would provide the required stability for a rigid body movement. In the present work we will present data that addresses these questions.