Novel Mechanism of Gating in the TrkH-TrkA Complex

Sunday, February 12, 2017 the viral transport from both the cargo and the ‘‘gate’’ (NPC) level in an unprecedented manner. 108-Plat Development of a Simultaneous Six-Color Fluorescence Microscope with Single-Molecule Sensitivity Jingyu Wang, Jamie Barnett, Luke Springall, Neil M. Kad. School of Biosciences, University of Kent, Canterbury, United Kingdom. Prokaryotic Nucleotide excision repair (NER) is a complex mechanism involving six proteins: UvrA, UvrB, UvrC, UvrD, DNA polymerase 1 and DNA ligase. To holistically study the kinetics of NER, multiple labelling and imaging channels are required to differentiate the parties involved in the damage search and repair activities. Using Quantum dot (Qdot)-conjugated UvrA, UvrB, UvrC, UvrD, DNA polymerase 1 and a Qdot labelled lesion we aim to detect and determine the significance of all the complexes that contribute to repair. For this purpose, we have adapted an existing fluorescence microscope into a multi-color simultaneous fluorescence microscope by spectrally separating 6 emission channels based on available Qdots: 525 nm, 565 nm, 585 nm, 625 nm, 655 nm and 705 nm. These six channels were created using parallel optical splitting devices distributed into two paths and imaged on two identical state-of-the-art Hamamatsu, ORCA-Flash4.0 V2 scientific CMOS cameras. Each low noise, high QE (>80%) camera sensor has 2048 x 2048 pixels, therefore every spectral channel has 682 x 2048 pixels; sufficiently large to image elongated DNA tightropes. Although the Qdots can be illuminated by a single 488 nm source, the fluorescence microscope also uses four excitation lasers: 405 nm, 488 nm, 561 nm and 637 nm combined into a single path. This allows for numerous combinations of channels to be excited and separately modulated using an Arduino-based control element linking the cameras to the lasers. Using this system, we present data obtained on the heterogeneity of the complexes formed during NER. 109-Plat Intracellular Delivery of Membrane Impermeable Photostable Fluorescent Probes into Living Cells for Super-Resolution Microscopy Yuji Ishitsuka1, Kai Wen Teng1, Pin Ren1, Yeoan Youn1, Xiang Deng2, Pinghua Ge1, Andrew Belmont2, Paul R. Selvin1. 1 Department of Physics, University of Illinois Urbana-Champaign, Urbana, IL, USA, 2Department of Cell and Developmental Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA. Specific labeling of the cellular target with fluorophore is one of the fundamental requirement in all fluorescence imaging techniques including single molecule and super-resolution microscopy techniques. While extracellular targets may be tagged with virtually any kind of probes, intracellular labeling of living cells is limited to the use of fluorescent proteins and limited selection of membrane permeable dyes. Here we show that pore forming proteins can be used to temporarily permeabilize the cells and allow delivery of various fluorescent probes, ranging from organic dyes (<1 kDa) to fluorescent immunoglobulin antibody (~150 kDa), for specific labeling of intracellular targets for live fluorescence cell imaging. We demonstrate that permeabilized cells can efficiently be recovered to carry on normal cellular processes shown by nuclear translocation of nanobody-labeled p65 proteins in response to chemical stimulation. One of the most photostable but membrane impermeable organic fluorophore, Atto 647N, has been delivered into cells to label actin fibers and kinesin dimers and to perform super-resolution fluorescence microscopy imaging dSTORM and single molecule tracking, respectively. This technique opens up a large number of stable, but otherwise membrane impermeable fluorescence labeling probes that are available for investigating transfected and endogenous intracellular targets. 110-Plat Bright and Photostable Fluorophores for Advanced Fluorescence Microscopy Qinsi Zheng1, Jonathan B. Grimm1, Anand K. Muthusamy1, Robert H. Singer1,2, Luke D. Lavis1. 1 Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA, 2Albert Einstein College of Medicine, New York, NY, USA. Advanced fluorescence microscopy, including single-molecule and superresolution imaging, demands bright and photostable fluorophores. We have recently reported a general approach to improve fluorophores brightness in living cells by substituting the N,N-dimethylamino groups found in classic dyes with four-membered azetidine rings (Nature Methods 12, 244250 (2015)). In an unpublished work we have synthesized new derivatives containing substituents on the azetidine ring. Using this approach we were able to fine tune the wavelength and fluorogenecity of the fluorophore without affecting brightness. Here, we report that several of these novel substituted-azetidine fluorophores, as well as the substituted-xanthene ones, exhibit substantial

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improvements in photostability in living cells. These fluorophores enable robust multi-color, wash-free imaging with a large photon budget. We are investigating their phototoxicity and mechanism in order to maximize the photostability, to generalize this approach to different fluorophores, and to apply these fluorophores to diverse biological settings, including living cells, tissues, and animals. We freely share our fluorophores with the academic community. This work is supported by HHMI and NIH (U01 EB 021236).

Platform: Membrane Protein Structures I 111-Plat Structure Inhibition and Regulation of a Two-Pore Channel TPC1 Alexander F. Kintzer, Robert M. Stroud. Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA. Membrane transport serves vital functions in the cell, providing nutrients for growth, relaying electrical signals, evading pathogens, and maintaining homeostasis. Voltage- and ligand-gated ion channels propagate cellular electrical signals by coupling changes in membrane potential and the binding of molecules to channel opening and selective passage of ions through the membrane barrier. Two-pore channels (TPCs) are intracellular ion channels that integrate changes in membrane potential, second messengers, and phosphorylation to control endolysosomal trafficking, autophagy, cellular ion and amino acid homeostasis, and ultra-long action potential-like signals. They broadly impact human diseases related to trafficking including filoviral infections, Parkinson’s disease, obesity, fatty liver disease, and Alzheimer’s disease. The response of TPCs to multiple cellular inputs suggests a multistate gating mechanism. Nevertheless, the mechanisms that govern cycles of activation and deactivation or ‘gating’ in the channel remain poorly understood. To understand the bases for ion permeation, channel activation, the location of voltage-sensing domains and regulatory ion-binding sites, and phosphoregulation, we determined the crystal structure of TPC1 from ˚ resolution. This reveals for the first time Arabidopsis thaliana to 2.87A how TPC channels assemble as ‘quasi-tetramers’ from two non-equivalent tandem pore-forming subunits. We determined sites of phosphorylation in the N-terminal and C-terminal domains that are positioned to allosterically modulate channel activation by cytoplasmic calcium. One of the two voltage sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal calcium locks VSD2 in a ‘resting’ conformation, distinct from the activated VSDs observed in structures of other voltage-gated ion channels. The structure shows how potent pharmacophore trans-Ned-19 allosterically acts to inhibit channel opening. In animals, trans-Ned-19 prevents infection by Ebola virus and Filoviruses by blocking fusion of the viral and endolysosomal membranes, thereby preventing delivery of their RNA into the host cytoplasm. The structure of TPC1 paves the way for understanding the complex function of these channels and may aid the development of antiviral compounds. 112-Plat Novel Mechanism of Gating in the TrkH-TrkA Complex Hanzhi Zhang1, Zhao Wang1,2, Mingqiang Rong1,3, Yaping Pan1, Wah Chiu1,2, Ming Zhou1. 1 Baylor College of Medicine, Houston, TX, USA, 2National Center for Macromolecular Imaging, Houston, TX, USA, 3Kunming Institute of Zoology, China Academic of Science, Kunming, China. The superfamily of Kþ transporters (SKT) is ubiquitous in bacteria, fungi and plants. SKT proteins are required for survival of bacteria in low Kþ conditions and are involved in salt regulation in fungi and plants. Bacterial SKTs have two components, a membrane embedded protein that resembles an ion channel and a cytosolic protein that regulates channel gating [1]. Crystal structures of two bacterial SKT systems were reported recently [2,3]. In both structures, the membrane embedded component forms a homodimer onto which a homotetrameric ring of the cytosolic protein docks. Single-channel activities of one of the complexes, the TrkH (membrane embedded) -TrkA (cytosolic) complex, were recorded and analyzed: ATP or ATP analogs such as AMPPNP activates the channel while ADP closes it [2]. The structure of the TrkH-TrkA complex is likely in a closed conformation because it was crystallized in the presence of NADH, a ligand that does not activates the channel. In order to understand how ATP or its analogs induces channel opening, we solved the structure of ˚ by x-ray crysthe TrkH-TrkA complex in the presence of AMPPNP to 3.29 A tallography. When compared to the previous structures, the new structure shows that each TrkA protomer binds to two AMPPNP molecules, and that the TrkA tetramer assumes an elongated conformation that likely induces a change in the TrkH. Conformational changes in the TrkH involve significant changes in the dimer interface and are different from any other channels of

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known structure. These new observations and hypotheses will be validated and tested by mutational and functional analyses. [1] Levin EJ, Zhou M. Recent progress on the structure and function of the TrkH/KtrB ion channel. Curr Opin Struct Biol. 2014;27:95-101. [2] Cao Y, Pan Y, Huang H, et al. Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature. 2013;496(7445):317-22. [3] Vieira-pires RS, Szollosi A, Morais-cabral JH. The structure of the KtrAB potassium transporter. Nature. 2013;496(7445):323-8. 113-Plat Structural and Functional Characterization of a Calcium-Activated Cation Channel From Tsukamurella Paurometabola Bala Dhakshnamoorthy, Ahmed Rohaim, Huan Rui, Lydia Blachowicz, Benoit Roux. University of Chicago, Chicago, IL, USA. The selectivity filter is an essential functional element of Kþ channels that is highly conserved both in terms of its primary sequence and its threedimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2þ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2þ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2þ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a preliminary characterization of this calcium-gated cation channel. 114-Plat Crystal Structure of a Low CO2-Inducible Protein, LCI1 in Chlamydomonas Reinhardtii Tsung-Han Chou. Physics and Astronomy, Iowa State University, Ames, IA, USA. The assimilation of atmospheric carbon dioxide (CO2) by microalgae and plants for photosynthesis has not been fully understood. Gaining insight into the intricate structural details of the CO2 scavenging mechanism by the photosynthetic green algae Chlamydomonas reinhardtii can pave the way for utilizing abundant CO2 as a valuable alternative fuel resource. Here, we present a crystal structure of the Chlamydomonas reinhardtii LCI1 channel, which is involved in the CO2-concentrating mechanism (CCM) to assimilate inorganic carbon resources. Combined with X-ray crystallography, mass spectrometry and computational simulation, our data indicate that the LCI1 membrane protein forms a trimeric assembly, in which each protomer conducts uncharged CO2 and shuttles this inorganic carbon species across the cell membrane. 115-Plat Structural Role of ABCG5/ABCG8 in Sterol Transport Jyh-Yeuan (Eric) Lee, Daniel Rosenbaum, Helen Hobbs. UT Southwestern Medical Center at Dallas, Dallas, TX, USA. ATP binding cassette (ABC) transporters play critical roles in maintaining sterol homeostasis in eukaryotic organisms, including yeast, plants and mammals. In humans, the heterodimeric ABCG5/ABCG8 (G5G8) mediates the excretion of cholesterol and dietary plant sterols into bile and into the gut lumen. Mutations inactivating either ABCG5 or ABCG8 cause sitosterolemia, a rare autosomal recessive genetic disorder characterized by plant sterol accumulation, hypercholesterolemia, and premature coronary atherosclerosis. ABCG5 and ABCG8 are half ABC transporters;each subunit consists of an N-terminal nucleotidebinding domain (NBD) and a C-terminal transmembrane domain (TMD). The NBDs dimerize to form two catalytically asymmetric nucleotide-binding sites (NBS), one that is catalytically active (NBS2) and the other inactive (NBS1). To understand the structural basis for G5G8-mediated sterol transport we developed a large-scale purification of human G5G8 by exploiting Pichia patoris yeast. We crystallized the transporter in lipid bilayers, solved its structure in a ˚ resolution, and generated the first atomic model nucleotide-free state at 3.9 A of an ABC sterol transporter. G5G8 presents a new structural configuration for the TMD of ABC transporters, which is present in a large and functionally diverse ABC2 superfamily. We discover that the TMD and the NBS are coupled through networks of interactions that differ between NBS1 and NBS2, reflecting the catalytic asymmetry of the transporter. A series of conserved polar residues in the TMD form polar networks that we proposed play a role in transmitting signals from the ATPase catalysis in the NBS to sterol transport on the TMD. Molecular dynamic simulation and long-range coevolution analysis revealed an inward-upward TMD movement that predicts a significant conformational change between the TMD subunits. Thus, the G5G8 structure provides a molecular framework that allows us to propose a mechanistic model for ABC

transporter-mediated sterol transport and to analyze the disruptive effects of mutations causing sitosterolemia. The structure will serve as a structural template for homology modelling to a wide range of transport system that is regulated by ABCG transporters and by ABC2 superfamily. 116-Plat Structural and Mechanistic Basis of Proton-Coupled Metal Ion Transport in the SLC11/NRAMP Family Cristina Manatschal, Ines A. Ehrnstorfer, Raimund Dutzler. Department of Biochemistry, University of Zurich, Zurich, Switzerland. Divalent metal ion transporters (DMTs) of the SLC11/NRAMP family transport iron and manganese across cellular membranes. These proteins are highly conserved across all kingdoms of life and thus likely share a common transport mechanism. Our previous crystal structure of Staphylococcus capitis DMT (ScaDMT) has established the structural relationship of the SLC11 family with the amino acid transporter LeuT and it revealed the location of a conserved transition-metal ion binding site in the center of the transporter. In this structure, ScaDMT adopts an inward-facing conformation. Recently, we have determined the crystal structure of Eremococcus coleocola DMT (EcoDMT) in an outward-facing conformation. Together these structures define the endpoints of the transport cycle. Functional assays with proteoliposomes established EcoDMT as secondary active transporter that couples the symport of Mn2þ and protons with a KM in the low micromolar range. Mutants of residues of the metal ion binding site severely affected both, Mn2þ and proton transport thus suggesting that the transport of protons requires conformational changes of the transporter. Inspection of both structures revealed two protonatable residues close to the metal ion binding site that have changed their accessibility to either side of the membrane as potential candidates for proton acceptors. Mutation of one of these residues, a conserved histidine on a-helix 6b, resulted in metal ion transport that appears to be no longer coupled to protons, which implies that this residue likely plays a central role in proton transport. Taken together, our studies have revealed the conformational changes underlying transition-metal ion transport in the SLC11 family by the alternate access mechanism and they provide important insights into the determinants of its coupling to protons. 117-Plat Structural Basis of Concentrative Nucleoside Transport Marsha M. Hirschi, Zachary L. Johnson, Seok-Yong Lee. Duke University, Durham, NC, USA. Nucleosides are essential molecules for the living cell. As precursors to nucleotides they serve to fuel the salvage pathway of DNA and RNA synthesis. Certain tissues, such as the brain and bone marrow, lack the capacity for de novo synthesis and therefore rely completely on the influx of nucleosides. Concentrative nucleoside transporters (CNTs) utilize sodium or proton gradients to transport nucleosides across the cell membrane. These secondary active transporters also play an essential role in the termination of adenosine signaling, which controls important cellular processes such as neuromodulation and cardiovascular function. In addition to natural substrates CNTs are also the conduit for many anti-cancer and anti-viral drugs, making them of special interest from a pharmacological point of view. Notably, CNTs are the main transport route for a popular pancreatic cancer drug, gemcitabine. We previously reported on the structure of CNT from Vibrio cholerae in complex with various substrates and nucleoside-like drugs. Each of these structures captured the transporter in the inward-facing occluded conformation. In order to describe the transport mechanism in further detail we performed crystallization studies with CNT from Neisseria wadsworthii (CNTNW). CNTNW is highly homologous to human CNT3, sharing 38% sequence identity and nearly identical substrate binding sites. Here we present crystal structures of CNTNW captured in alternative conformations. We confirmed the physiological relevance of the conformations by crosslinking experiments. Our structural analyses and functional studies provide new insights into the mechanism of CNTs and cellular nucleoside uptake in molecular detail. 118-Plat Investigating the Structure of the Drug Transporter EmrE Maureen Leninger, James R. Banigan, Geliana Abramov, Nathaniel J. Traaseth. Chemistry, New York University, New York, NY, USA. Multidrug resistance in bacteria is a critical challenge in public health and drug discovery. One of the primary mechanisms of resistance is efflux pumps, which couple an energetically favorable process with the export of a drug molecule against its concentration gradient.1 Efflux pumps from the small multidrug resistance protein family are ubiquitous among bacteria. These secondary active transporters couple the efflux of a wide variety of toxic compounds with the proton gradient of the inner membrane.2 To gain insight into this