Impact of Mutations on the Structure of the Human Potassium Channel KCNQ1

Sunday, February 28, 2016 Anaerobic gut fungi reside in the digestive tract of large herbivores where they enable the digestion of resilient plant biomass into fermentable sugars. It is likely that the membrane envelope of these important but woefully understudied organisms is involved in their cellulolytic lifestyle. Our studies suggest that these fungal membranes contain a number of sugar transporters and sensors that are potentially valuable tools for the biotech community. Characterization of these entities will also shed light on the remarkable abilities of these most early diverging eukaryotes. Here, we have used RNA-Seq to study the membrane transcriptome of three strains of gut fungi: Anaeromyces sp. S4, Piromyces sp. finn, and Neocallimastix sp. G1 at high resolution. Hydropathy analyses suggest that at least 20% of the transcripts in each strain encode proteins that are integral membrane proteins. Among these are transporters and proteins involved in energy metabolism and signaling. Surprisingly, we find a number of membrane-anchored proteins that are homologous to bacterial sugarbinding proteins. Some of these putative sugar-binding domains are fused to class 3 G-protein coupled receptors (GPCRs), and as such suggest that GPCRs play a sugar-sensing role in primitive fungi. 309-Pos Board B89 Preparation and Delivery of Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography Andrii Ishchenko1, Vadim Cherezov1, Wei Liu2. 1 BRIDGE Institute at University of Southern California, Los Angeles, CA, USA, 2Arizona State University, Tempe, AZ, USA. Membrane proteins (MPs) are important components of cellular membranes and primary drug targets. Rational drug design relies on the exact structural information about the protein, however MPs are difficult to handle and crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization method, which yields well-diffracting but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. Introduction of the X-ray free-electron laser (XFEL) source that produces extremely bright femtosecond pulses enabled room temperature data collection from microcrystals with minimal, if any, radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCPSFX) have resulted in high-resolution structures of several human G proteincoupled receptors, the largest and the most important drug target superfamily, yet notoriously difficult for structural determination. In LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of membrane protein microcrystals to the intersection with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can significantly improve the diffraction resolution when only micron-sized crystals are available and when crystal quality is too poor to yield interpretable structural data at conventional synchrotron microfocus beamlines. Here we present the protocol for preparation, characterization and delivery of microcrystals in LCP for LCPSFX experiments. 310-Pos Board B90 Impact of Mutations on the Structure of the Human Potassium Channel KCNQ1 Hui Huang1, Keenan C. Taylor1, Brett M. Kroncke1, Alfred L. George2, Charles R. Sanders1. 1 Biochemistry, Vanderbilt University, Nashville, TN, USA, 2Pharmacology, Northwestern University, Chicago, IL, USA. Approximately one in 2000 newborns are affected by congenital long QT syndrome (LQTS), which is a life-threatening cardiac disorder. Mutations in the voltage-gated potassium channel KCNQ1 and its accessory protein KCNE1 cause 50% of congenital LQTS. Up to now, more than 400 mutations have been identified in KCNQ1 from LQTS subjects. The mechanistic effects of many of these mutations remain unknown. In the present study, we have investigated the specific effects of 32 KCNQ1 mutations on the structural properties of isolated voltage-sensor domain of KCNQ1 using solution nuclear magnetic resonance spectroscopy. The 32 mutations are disease causing, benign, or of unknown significance. The high quality NMR spectra of wild type and of one engineered double mutation E160R-S225E serve as reference spectra, representing the open state (active conformation) and closed state (inactive conformation) forms of the channel, respectively. Mutants were labeled with 15N and purified into lysomyristoylphosphatidylglycerol (LMPG) detergent micelles. The 1H-15N TROSY-heteronuclear single quantum coherence spectroscopy spectrum of each mutant was collected and compared with the reference spectra. The effect of each mutation on the equilibrium between the channel active conformation and inactive conformation was determined. These results will advance our understanding of the molecular basis of LQTS pathogenesis and will be important for the rational design of anti-arrhythmia therapeutics for patients harboring KCNQ1 mutations.

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311-Pos Board B91 Characterizing the Structural Basis for USHER Activation Natalie S. Omattage1, Zengqin Deng2, Peng Yuan2, Scott J. Hultgren1. 1 Molecular Microbiology & Microbial Pathogenesis, Washington University in St. Louis, St. Louis, MO, USA, 2Cell Biology and Physiology, Washington University in St. Louis, St. Louis, MO, USA. Uropathogenic Escherichia coli (UPEC) is the causative agent of 85% of community-acquired urinary tract infection. UPEC utilize the type 1 pilus to mediate adhesion, via its tip-located FimH adhesin, to mannosylated receptors on the surface of bladder epithelium. Type 1 pilus assembly is an intricate molecular process that requires the passage and polymerization of hundreds of subunits across both the inner and outer membranes. The outer membrane usher, FimD, serves as an assembly platform to catalyze subunit polymerization. FimD is comprised of five functional domains: a 24-stranded transmembrane ß-barrel translocation domain (TD), a ß-sandwich plug domain (PD), a periplasmic N-terminal domain (NTD), and two periplasmic C-terminal domains (CTD1 and 2). In the absence of pilus assembly, the PD is located within the TD lumen, blocking the TD pore. The high-affinity binding of the chaperone-adhesin complex (FimCH) to the FimD NTD initiates pilus assembly with: i) movement of the PD from the TD lumen to the periplasm; ii) rearrangement of the TD ß-barrel from an oval-shaped to a large circular pore; and iii) the formation of a stable interaction between the PD and the NTD. These marked conformational changes prime the TD for subunit-subunit polymerization and transit of the growing pilus fiber through its pore. In order to understand how binding at the NTD induces PD movement, it is crucial to obtain structures of the full-length usher alone and with its NTD in complex with FimCH. We have developed systems for expression and purification of FimD, and for reconstitution of FimDCH ternary complex in vitro. Currently, we are utilizing these tools to obtain the structures of fulllength apo-FimD and FimDCH. These structures will provide the first insight into the molecular cues necessary for maintaining the equilibrium between an inactive and active usher state. 312-Pos Board B92 Protocol to Avoid Possible Artifacts in Atomistic Simulation of GPCR Proteins whose Crystal Structure is Heavily Engineered Moutusi Manna1, Waldemar Kulig1, Matti Javanainen1, Joona Tynkkynen1, Ulf Hensen2, Daniel J. Mu¨ller2, Tomasz Rog1, Ilpo Vattulainen1,3. 1 Department of Physics, Tampere University of Technology, Tampere, Finland, 2Department of Biosystems Science and Engineering (D-BSSE), ETH-Zu¨rich, Basel, Switzerland, 3MEMPHYS-Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark. G protein-coupled receptors (GPCRs) are versatile signaling proteins that mediate diverse cellular responses. Atomistic molecular dynamics (MD) simulations are widely used to elucidate the properties of GPCRs. These simulations are based on three-dimensional protein structures that in turn are often based on crystallography. However, to facilitate structure determination, typically the crystallized proteins are heavily engineered, including structural modifications (mutations, replacement of protein sequences by antibodies, bound ligands, etc.) whose impact on protein structure and dynamics is largely unknown. Here we address this issue through atomistic MD simulations, focusing on the b2-adrenergic receptor (b2AR), a well-characterized GPCR. Starting from an inactive-state crystal structure of b2AR, we reverted the numerous structural modifications done on b2AR in multiple consecutive steps, one at a time, each followed by extensive equilibration in a lipid membrane. The systematic step-by-step approach provides results that are superior in terms of maintaining protein structural stability, as compared to the usual and computationally less expensive approach of removing all modifications instantaneously at once. Another great advantage of the step-wise method is that it clarifies the effects of individual crystallization modifications on the native properties of the receptor, such as on the dynamics of the ligand and the G-protein binding sites and the packing at the transmembrane helix interface of b2AR in the present case. Our results emphasize that the preparation of membrane protein structure for atomistic MD simulations is a very delicate and sensitive process and can lead to significant artifacts if done in a straightforward manner by cutting corners. We consider that the protocol described here (step-by-step approach) may offer a useful strategy for simulating a variety of native state GPCRs, whose crystal structures suffer from similar structural engineering. 313-Pos Board B93 Dynamical and Structural Alterations withing Lipid-Protein Assemblies Control Apoptotic Pore Formation - A Solid State NMR Study Artur P.G. Dingeldein, Martin Lidman, Tobias Sparrman, Gerhard Gro¨bner. Chemistry, Umea˚ University, Umea˚, Sweden. Not only provide mitochondria the majority of ATP supplies, but also play an important role in regulated cell death. The regulator proteins deciding upon the