Day 1 :
Keynote Forum
Jeffrey Skolnick
Georgia Instiute of Technology, USA
Keynote: Comprehensive prediction of drug-protein interactions and side effects for the human proteome
Time : 09:00-09:40
Biography:
Jeffrey Skolnick is the Director of the Center for the Study of Systems Biology in the School of Biology at the Georgia Institute of Technology. He is also the Mary and Maisie Gibson Chair & Georgia Research Alliance Eminent Scholar in Computational Systems Biology. Among his awards is the SURA 2014 Distinguished Scientist Award, an Alfred P. Sloan Research Fellowship and he is a Fellow of the American Association for the Advancement of Science and the Biophysical Society. He is the author of over 355 publications, has an h-index of 76, and has served on numerous editorial boards.
Abstract:
Identifying unexpected drug-protein interactions is crucial for drug repurposing. We develop a comprehensive proteome scale approach that predicts human protein targets and side effect of drugs. For drug-protein interaction prediction, FINDSITEcomb, whose average precision is ~30% and recall ~27%, is employed. For side effect prediction, a new method is developed with a precision of ~57% and a recall of ~24%. Our predictions show that drugs are quite promiscuous, with the average (median) number of human targets per drug of 329 (38), while a given protein interacts with 57 drugs. The result implies that drug side effects are inevitable and existing drugs may be useful for repurposing, with only ~1,000 human proteins likely causing serious side effects. A killing index derived from serious side effects has a strong correlation with FDA approved drugs being withdrawn. Therefore, it provides a pre-filter for new drug development. The methodology is free to the academic community on the DR. PRODIS (DRugome, PROteome, and DISeasome) webserver at http://cssb.biology.gatech.edu/dr.prodis/. DR. PRODIS provides protein targets of drugs, drugs for a given protein target, associated diseases and side effects of drugs, as well as an interface for the virtual target screening of new compounds. Successful applications of the methodology to treat Chronic Fatigue Syndrome, to identify novel antibiotic leads and promising anti-seizure drugs are described.
Keynote Forum
Michael G Rossmann
Professor, Purdue University, USA
Keynote: A personal history of structural virology
Time : 13:30-14:10
Biography:
Michael Rossmann graduated from the University of London (B.Sc. General in Physics and Mathematics, 1950; B.Sc. Special 1951 in Physics; M.Sc., 1953) and the University of Glasgow (Ph.D., 1956). He was a postdoctoral fellow with Prof. William Lipscomb at the University of Minnesota (1956-1958) and a Research Associate with Dr. Max Perutz at the MRC Laboratory of Molecular Biology, Cambridge, England (1958-1964). Currently he is the Hanley Professor of Biological Sciences at Purdue University, where he has worked for the last 52 years. His laboratory utilizes X-ray crystallography and electron microscopy to study the structures of animal and bacterial viruses.
Abstract:
As a graduate student in Glasgow and post-doc in Minnesota I acquired a fairly good understanding of structural crystallography and the use of very early electronic computers. I then joined Max Perutz’s laboratory in 1958 as a post-doc and was a member of the small team who witnessed the determination of the first 3D protein structures. I then started my own laboratory at Purdue University in 1964 with the aim of determining the atomic structure of a virus. As a start I worked on dehydrogenases that were oligomeric as were also viruses. That led to the recognition of the fold capable of binding nucleotides. This gave enough confidence to look at the structure of a plant virus. Steve Harrison at Harvard was also working on the structure of a plant virus, leading to the first two virus structures at near-atomic resolution in about 1979. From there we looked at the structure of a common cold virus (1985), the first animal virus to be determined. Many other viruses followed in our lab and many other labs, giving a wealth of biological understanding. In the current year we published the structure of Zika virus, using electron microscopy instead of X-ray crystallography.
- Symposium on New technologies in structural studies y cryoEM
Chair
Qiu-Xing Jiang
University of Florida, USA
Co-Chair
Kenneth A Taylor
Florida State University, USA
Session Introduction
Michael G Rossmann
Purdue University, USA
Title: The structure of Zika virus
Time : 09:40-10:05
Biography:
Michael Rossmann graduated from the University of London (B.Sc. General in Physics and Mathematics, 1950; B.Sc. Special 1951 in Physics; M.Sc., 1953) and the University of Glasgow (Ph.D., 1956). He was a postdoctoral fellow with Prof. William Lipscomb at the University of Minnesota (1956-1958) and a Research Associate with Dr. Max Perutz at the MRC Laboratory of Molecular Biology, Cambridge, England (1958-1964). Currently he is the Hanley Professor of Biological Sciences at Purdue University, where he has worked for the last 52 years. His laboratory utilizes X-ray crystallography and electron microscopy to study the structures of animal and bacterial viruses.
Abstract:
Zika virus belongs to the flavivirus family that includes dengue virus, West Nile virus, yellow fever virus and other human pathogens. The structure of dengue virus was the first flavivirus to be determined. It was accomplished by obtaining a low resolution image of the whole virus by cryoEM and determining the structure of the major capsid protein of the homologous tick-borne encephalitis virus crystallographically. When Zika started to be recognized as a major threat to humanity, cryoEM had advanced to allow us to determine the structure of Zika virus to near-atomic resolution using only cryoEM. The most important difference between dengue and Zika virus was found to be at a glycosylation site which is suspected to be involved in the recognition of molecules on the surface of potential host cells.
Liguo Wang
University of Washington, USA
Title: Random Spherically Constrained (RSC) single particle cryo-EM for structures of membrane proteins in a membrane environment
Time : 10:05-10:30
Biography:
Liguo Wang completed his PhD at the age of 33 years from Cornell University and his postdoctoral studies from Yale University School of Medicine. He is an assistant professor in the department of Biological Structure at the University of Washington. He has more than 10 years of expertise in cryo-Electron Microscopy, and developed a method to study structures of membrane proteins in lipid environments, which make it possible to manipulate the functional states of membrane proteins. He published more than 20 papers in reputed journals. He also served as a reviewer for several high-ranking journals and a reviewer of scientific proposals for a few scientific organizations.
Abstract:
Recently, a steadily growing community of researchers has been attracted by the capability of cryo-Electron Microscopy (cryo-EM) to obtain atomic-resolution structures without the need to crystalize their samples. To study membrane protein structures using cryo-EM, membrane proteins are usually extracted from cell membranes and dissolved in detergents. However, both functional and structural studies clearly highlight the importance of a lipid membrane environment to preserve protein integrity and activity. To restore the lipid membrane environment of membrane proteins, I have been developing a platform, called “random spherically constrained” (RSC) single-particle cryo-EM, for both structural and functional studies of membrane proteins. The RSC platform establishes the lipid environment for membrane proteins, and makes it possible, for the first time, to apply desired transmembrane potential to trap voltage-gated ion channels in desired functional states for structural analysis. To confirm the absolute amplitude of the transmembrane potential, the hyperpolarization- activated cyclic nucleotide-gated (HCN2) channel was investigated as a model protein, as it only opens at very negative transmembrane potentials. The results confirmed that a negative potential of -120 mV was successfully established as predicted. The RSC method has been successfully employed to obtain the structures of both the large conductance voltage- and calcium-activated potassium (BK) and HCN2 channels in lipid membranes. This confirmed that the RSC method could be used to manipulate the functional states of other voltage-gated ion channels, which is critical for understanding the mechanism under which the ion channel responds to the transmembrane potential.
Qiu-Xing Jiang
University of Florida, USA
Title: ChemiC: Chemically functionalized nanometer-thick carbon films for cryoEM studies of biological complexes
Time : 10:30-10:55
Biography:
Jiang obtained his PhD from Yale University and did his postdoctoral training at Rockefeller University. He is currently an Associate Professor in UF and serves as the Faculty Director of Electron Microscopy (EM) at the UF Interdisciplinary Center for Biotechology Research, where EM is used for revealing the structural basis of biological machineries. He has published more than 25 papers in reputed journals. His lab focuses on structural and functional studies of intracellular RNA-binding complexes and ion channels invovled in membrane excitability and regulated secretion, and has been developing new methods for cryoEM imaging of single molecules.
Abstract:
With recent successful development of maximum-likelihood based refinement algorithms, direct electron detectors and motion correction, phase plates, energy filters and automatic data collection and particle selection, preparation of good cryoEM specimens and reliable analysis of conformational and/or compositional heterogeneity in the images of single molecular complexes are becoming the rate-limiting steps in cryoEM studies of biological specimens. ChemiC was developed in my laboratory to facilitate selective enrichment of nanogram amounts of biological complexes to the surfaces of chemically functionalized carbon-films, which, compared to the conventional, physically-treated carbon films, has multiple advantages. It allows affinity-based selection without introducing a significant amount of biomass, retains all attached molecules in a thin layer of vitrified ice that is less than 40 nm thick and has much less ice-scattering noise than conventional 100-150 nm thick ice layers, and enables controlled assembly of specific biological processes or synchronization of biological complexes in a targeted conformational state. This new method has been used for different projects. I will discuss the ChemiC technology and its applications in cryoEM specimen preparation and imaging and will present the recent progress in further development of the technology for different purposes.
Kenneth A Taylor
Florida State University, USA
Title: Deciphering intermolecular interactions in complex heterogeneous systems
Time : 11:15-11:40
Biography:
Kenneth A Taylor has completed his PhD in 1975 from University of California Berkeley on the topic of Electron Microscopy and Electron Diffraction of Frozen Hydrated Biological Specimens, widely regarded at the start of the field of cryo-electron microscopy. He has spent 4 years as a Post doctorate at the MRC Laboratory of Molecular Biology and 15 years on the faculty at Duke University Medical Center. He is currently the Donald L. D. Caspar Professor of Biological Science. He studies the structure of macromolecular assemblies in muscle, the cytoskeleton and viruses using cryo-electron microscopy and cryo-electron tomography.
Abstract:
Many biological systems contain complex mixtures of filaments, such as actin containing and myosin containing filaments and the cytoskeleton. The properties of the interacting partners are keys to understanding how the systems function. In muscle, the filament systems are composed of multiple proteins that modify the basic filament. In the case of actin, the troponin-tropomyosin system provides a mechanism for regulating attachment by the myosin motors and thus muscle contraction. During muscle contraction, myosin motors attach the thin filament in multiple ways producing a heterogeneous system that is difficult to dissect. Many proteins interact with F-actin to form cross-linked bundles. Among these are fimbrin, villin, α-actinin and several glycolytic enzymes and even elevated concentrations of magnesium and polylysine. An important characteristic of these cross-linkers is an ability to form 2-D rafts on positively charged lipid monolayers. Actin rafts should be ideal for structure determination by electron microscopy (EM) but several problems exist, in particular heterogeneity. All raft cross-linkers produce polymorphic rafts that are either polar or have mixed polarity. Even bundles of defined polarity can have heterogeneous cross linkers. We have developed a strategy for obtaining information from these heterogeneous systems using sub-volume alignment and classification that will be applicable to this type of problem and perhaps many others where there is difficulty obtaining large populations of homogeneous entities, be they filament segments or single particles.
Matthew T Swulius
California Institute of Technology, USA
Title: Breathing life into static images: Combining electron cryotomography and coarse-grained simulations to explore mechanisms of eukaryotic cell division machinery in vivo
Time : 11:40-12:05
Biography:
Matthew T Swulius has completed his PhD at University of Texas Health Science Center at Houston in 2010, where he studied structural and protein compositional changes in the synapse throughout development. He is currently a senior Post-doc in Grant Jensen’s lab at Caltech, where he has contributed to the field of bacterial cell biology by studying the MreB cytoskeleton using electron cryotomography and to eukaryotic cell division using cryo-focused ion beam milling and correlated light and electron microscopy to image the native structure of the actomyosin ring in fission yeast.
Abstract:
While electron cryotomography (ECT) is a powerful technique for imaging unique biological structures to molecular resolution, some protein machinery is complex and highly dynamic, making static images difficult to interpret on their own. In such cases, coarse-grained simulations can be employed in order to interpret and test the mechanistic implications of such experimental data. Here we use a combination of ECT and simulation to study the structure and contractile mechanism of the actomyosin ring (AMR) in fission yeast. Because fission yeast are beyond the thickness amenable to cryoEM, we used cryo-focused ion beam milling and cryosectioning to gain access to natively preserved AMRs. ECT revealed bundles of actin filaments running parallel to one another that were “saddling” the leading edge of the septum, and no direct contact between actin and the membrane was observed, refuting the notion that F-actin is connected to the membrane at cytokineteic nodes. After exploring a variety of actomyosin configurations by 3D coarse-grained simulation, we propose a model that best agrees with our and other published experimental data.
Youdong Jack Mao
Harvard Medical School, USA
Title: Advanced machine-learning algorithms for improving high-performance cryo-EM data analysis
Time : 12:05-12:30
Biography:
Youdong Mao has obtained his PhD in Physics from Peking University in 2005 and completed his Postdoctoral training in Biophysics at Harvard Medical School in 2012. He has then worked at the Faculty of Dana-Farber Cancer Institute, Harvard Medical School. In 2015, he has joined the Faculty at the School of Physics and Center for Quantitative Biology at Peking University. His research interest lies in developing multidisciplinary approaches across the areas of structural biology, electron microscopy and high-performance machine learning.
Abstract:
Machine learning technology represents an intriguing avenue in the methodology development for cryo-EM structure determination. In this presentation, we explore two aspects of machine learning development that help advance cryo-EM data analysis. First, structural heterogeneity in single-particle images presents a major challenge for high-resolution cryo-EM structure determination. Recently we introduce a statistical manifold learning approach for unsupervised single-particle deep classification. When optimized for Intel High-Performance Computing (HPC) processors, our approach implemented in ROME software package, can generate thousands of reference-free class averages within several hours from hundreds of thousands of single-particle cryo-EM images. Deep classification thus assists in computational purification of single-particle datasets for high-resolution reconstruction. Second, particle extraction represents a major practical bottleneck in the structure determination of biological macromolecular complexes by single-particle cryo-EM. We developed a deep learning-based algorithmic framework, DeepEM, for single-particle recognition from noisy cryo-EM micrographs, enabling automated particle picking, selection and verification in an integrated fashion. Our approach exhibits improved performance and high accuracy when tested on the standard KLH dataset as well as several challenging experimental cryo-EM datasets.
- Track 1: Structural Biology | Track 2: 3D Structure Determination | Track 3: Computational Approaches
Location:
Chair
Fumio Hirata
Toyota Physical & Chemical Research Institute, Japan
Co-Chair
John H Miller
Victoria University of Wellington
Session Introduction
John H. Miller
Victoria University of Wellington, New Zealand
Title: Tubulin structural interactions with small molecule microtubule-stabilizing agents
Time : 13:55-14:15
Biography:
John Miller completed his PhD in biological sciences in 1971 from Stanford University, carried out his postdoctoral research in molecular biology and physiology and at the University of California, San Diego, and then accepted a lectureship at Victoria University of Wellington in 1977. He was promoted to full professor in 2008. He teaches courses in mammalian physiology, cell biology, and development. His research interests are in the mode of action of novel natural products and their development as anticancer agents. He has published more than 113 papers in peer-reviewed journals and has written 4 book chapters.
Abstract:
Tubulin, a 50 kDa protein, associates into α, β-heterodimers which polymerize into the microtubule, a major cytoskeletal component of all eukaryote cells. Microtubules consist of 13 protofilaments with the α,β-dimers stacked head-to-tail. Numerous molecules, both endogenous and exogenous, bind to tubulin and affect its ability to polymerize and depolymerize. Insights into the structure of tubulin and the binding sites of different ligands were first obtained from electron crystallography of Zn- and paclitaxel-stabilized, antiparallel sheets. Paclitaxel is the first known microtubule-stabilizing agent. More recent studies have used a T2R complex consisting of a stathmin-like domain bound to two heterodimers linked in a head-to-tail fashion. Stathmin, by inducing a curved dimer structure, prevents its assembly into polymers. This T2R complex, often in combination with the enzyme tubulin tyrosine ligase has allowed detailed structural mapping by X- ray crystallography to less than 2.3 Å resolution. There are two known microtubule-destabilizing sites on β-tubulin, the colchicine site and the vinca alkaloid site, and two stabilizing sites, the taxoid site and the laulimalide/peloruside site. Recent X-ray crystallography analysis has confirmed the location of the stabilizing sites and shown that ligands that bind the two different sites can bind simultaneously. Adding both types of ligands together can lead to synergistic effects on the dynamicity of the microtubules, both in solution and inside cells; however, the mechanisms of stabilization and synergy between ligands are not fully understood. In collaboration with Eva Nogales at Berkeley, high resolution cryo-EM studies are currently underway on the structural association between peloruside and tubulin.
Kazuhiro Ogata
Yokohama City University Graduate School of Medicine, Japan
Title: Molecular behavior of a higher-order complex of multiple transcription factors on enhancer site upon phosphorylation
Time : 14:15-14:35
Biography:
K. Ogata has completed his PhD at the age of 27 years from Yokohama City University in 1992 and postdoctoral studies from RIKEN tsukuba institute. In 1997, He became the leader of the regulation of protein function project, Kanagawa Academy of Science and Technology (KAST), and from 2001, the professor and chairperson of Department of Biochemistry, Yokohama City University Graduate School of Medicine. He has published papers in JMB, PNAS, NSMB, and Cell, and had been serving as an editorial board member of BBRC (2009-2010)
Abstract:
Transcriptional regulation is a fundamental mechanism for cell proliferation and differentiation. A major player in this process is a member of tanscription factors (TFs). Activities of TFs are modulated by chemical modifications such as phosphorylation through cell signaling pathways directing cell functions. Molecular mechanisms of regulation for the functional high-order assembly consisting of multiple TFs and enhancer DNA (enhanceosome) under cell signaling are largely unknown. So far, we investigated changes in assembly state of an enhanceosome upon a TF’s phosphorylation, using crystallographical, biophysical, and molecular dynamical analyses. Here, we will illustrate the case of Ets1 in non-phosphorylated and phosphorylated forms on its target gene enhancers with or without its partner TFs, Runx1/CBF or Pax5, as an example. A structure-function relationship of a TF-DNA assembly and an effect by phosphorylation will be discussed.
Fumio Hirata
Toyota Physical & Chemical Research Institute, Japan
Title: Structure, fluctuation, and function of biomolecules in solution explored by the 3D-RISM/RISM theory
Time : 14:35-14:55
Biography:
Fumio Hirata has completed his PhD from Hokkaido University and postdoctoral studies from State be University of New York, University of Texas, and Rutgers University. He was an associate professor in Kyoto University, and a professor in Institute for Molecular Science. He is currently a professor emeritus of the institute. He has published more than 200 papers in reputed journals and has served as an editorial board member of repute.
Abstract:
There are two physicochemical processes which are essential for living bodies to maintain their life: “self-organization” and “molecular recognition.” Protein folding and formation of cell-membrane are typical examples of the former process, in which biomolecules have to overcome the entropy barrier to organize themselves into some characteristic structure. On the other hand, a molecular recognition process concerns whenever a biomolecule performs its function as a “molecular-machine.” For examples, in order for the enzymatic reaction to occur, substrate molecules should be accommodated first by the protein in its reaction pocket to form so-called an enzyme–substrate (ES) complex. The two processes may not proceed spontaneously if biomolecules and ligand molecules are exisiting by themselves in “vacuum,” because those are not in favor with respect to entropy. For instance, the protein folding is a process in which a protein folds into a native conformation, the state of least entropy, from the random coil, the state of largest entropy. Then, why do those processes occur spontaneously in our body? It is because there is always “aqueous solution” in the real environment of a living body.
Toshiya Senda
High Energy Accelerator Research Organization, Japan
Title: Discovery of a GTP sensor using a structural reverse genetic approach
Time : 14:55-15:15
Biography:
Toshiya Senda has completed his PhD from Nagaoka University of Technology (Niigata, Japan) in 1995. He was a research associate in Nagaoka University of Technology (1995-2001) and a senior researcher in Institute of Advanced Industrial Science and Technology (2001-2012). Now, he is the director/professor of Structural Biology Research Center of High Energy Accelerator Research Organization in JAPAN. He was awarded the CrSJ (Crystallographic society of Japan) award in 2014 (Structural biology studies of CagA from Helicobacter pylori and histone chaperon CIA/ASF1).
Abstract:
GTP is an energy molecule in the cell and required in protein synthesis. Reduction of GTP concentration results in slow cell growth; inversely, rapidly growing cells have elevated GTP concentration. Because of its vital roles in cell growth, GTP concentration should be monitored and homeostatically regulated in the cell. However, a sensing mechanism of cellular GTP concentration remains elusive. Here, we show that a lipid kinase, PI5P4Kβ, serves as a GTP sensor and GTP concentration functions as a metabolic cue via PI5P4Kβ. Our proteomics and biochemical study revealed that PI5P4Kβ binds GTP and its enzyme activity is significantly higher with GTP than with ATP; PI5P4Kβ mainly utilizes GTP for phosphorylation of PI5P. Furthermore, the kinetic characters of PI5P4Kβ are suitable to detect the change of cellular GTP concentration. These biochemical characteristics suggested that PI5P4Kβ is a GTP sensor in the cell. However, since PI5P4Kβ can utilize not only GTP but also ATP for the enzyme reaction in the cell, a simple knock out/down experiment is insufficient to analyze the biological function of the GTP-sensing activity of PI5P4Kβ. We therefore took a structural reverse genetic approach. First, we determined crystal structures of PI5P4Kβ-ATP/GTP complexes and used the crystal structures to prepare a PI5P4Kβ mutant that lacks GTP-sensing activity without changing ATP-dependent activity. We then preformed biological and metabolomic analyses with the PI5P4Kβ mutant, revealing that PI5P4Kβ serves as a GTP-sensor. The GTP-sensing activity of PI5P4Kβ is critical for metabolic adaptation and tumorigenesis.
Shigeyuki Yokoyama
RIKEN Structural Biology Laboratory, Japan
Title: Structural and synthetic biology of aminoacyl-tRNA synthesis and translation
Time : 15:15-15:35
Biography:
Shigeyuki Yokoyama has obtained his PhD degree from The University of Tokyo in 1981. He was an Associate Professor (1986-1991) and Professor (1991-2012) at The University of Tokyo and presently an Emeritus Professor. He was also appointed as the Director of the Cellular Signaling Laboratory (1993-2004), the Structural Molecular Biology Laboratory (2004-2006), the Protein Research Group (1998-2008) and the Systems and Structural Biology Center (2008-2013). He is a Distinguished Senior Scientist and directing the Structural Biology Laboratory at RIKEN. He has published more than 800 papers and has been serving as Editorial Board Members of Nucleic Acids Research etc.
Abstract:
In the protein synthesis according to the genetic code, each of the twenty amino acids is provided as an aminoacyl-tRNA. We have been studying the mechanisms of the specific aminoacyl-tRNA synthesis by crystallography and mutagenesis. Aminoacyl-tRNA synthetases (aaRSs) specifically recognize their substrate amino acid and tRNA species. The 3’-CCA region, conserved in all tRNAs, is single stranded and flexible, of which the terminal adenosine is aminoacylated by the aminoacylation domain of aaRS. The major recognition element, e.g., the anticodon of a tRNA interacts with the site on the tRNA recognition domain, far from the aminoacylation catalytic site. Several aaRSs have the editing domain which hydrolyzes incorrectly formed aminoacyl-tRNAs and the terminal adenosine shuttles between the two catalytic sites. For amino acids such as glutamine and selenocysteine, their precursor amino acids are attached to their tRNAs by others’ aaRSs and then enzymatically converted to the correct ones. We found that, in most of these processes of the tRNA and amino acid selection, the flexibility of the CCA region of tRNA is important. On the structural basis, we have engineered the substrate specificities of tyrosiyl, phosphoseryl and pyrrolysyl-tRNA synthetases for expansion of the genetic code to incorporate various unnatural amino acids site specifically into proteins in mammalian cells and in the Escherichia coli protein synthesis in vivo and in vitro. We used the expanded genetic code to produce proteins with epigenetic modifications, drug conjugation or fluorescence probes and to photocrosslink protein complexes including membrane protein complexes in vitro and/or in vivo.
Lei Zhang
Xi’an Jiaotong University, China
Title: Three dimensional dynamic structrues of DNA-nanogold conjugates by individidual-particle electron tomography
Time : 15:20-15:40
Biography:
Prof. Lei Zhang has completed his PhD at the age of 29 years from Xi’an Jiaotong University and postdoctoral studies from Lawrence Berkeley National Laboratory. He has published more than 30 papers in reputed journals and has been serving as an editorial board member of several journals. His researches focus on the determination of protein structure and functions by advanced electron microscpies.
Abstract:
DNA base-pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of the three-dimensional (3D) structure of the DNA-nanocrystal hybridized building blocks. Existing techniques do not allow for the structural determination of these flexible and heterogeneous samples. Here, we employed electron tomography and negative-staining techniques to investigate the 3D structure of DNA-nanogold conjugates that were self-assembled from a mixture of an 84-base-pair double-stranded DNA (dsDNA) conjugated with two 5-nm nanogold particles for potential substrates in plasmon coupling experiments. We reconstructed 14 electron density maps at a resolution of ~2 nm from each individual dsDNA-nanogold particle using the individual-particle electron tomography (IPET) reconstruction method. Using these 3D density maps as a constraint, we projected a standard flexible DNA model onto the observed EM density maps and derived 14 conformations of dsDNA by molecular dynamics simulations. The variation of the conformations was consistent with the variation from liquid solution, but the IPET approach provides the most complete experimental determination of the flexibility and fluctuation range of these directed nanocrystal assemblies to date. The general features revealed by these experiments can be expected to occur in a broad range of DNA-assembled nanostructures.
- Track 4: Signalling Biology | Track 5: Drug Designing | Track 6: Sequencing
Chair
Takeshi Kikuchi
Ritsumeikan University, Japan
Co-Chair
Elizabeth J Goldsmith
University of Texas Southwestern Medical Center at Dallas, USA
Session Introduction
Seth Pincus
Children’s Hospital and LSU School of Medicine, USA
Title: Development of anti-HIV double variable domain antibodies that bind both gp120 and gp41 on the envelope protein
Time : 15:50-16:10
Biography:
Seth Pincus received his M.D. from New York University. Following a Pediatrics residency at the University of Utah, he trained in the Immunology Branch of NCI. His first faculty appointment was at the University of Utah, followed by stints at the NIAID Rocky Mountain Labs, at Montana State University where he Chaired the Department of Microbiology, and from there to his current position in the Departments of Microbiology and Pediatrics, at LSU School of Medicine, New Orleans. He has published over 100 peer-reveiwed papers, textbook chapters, and invited articles. He has served on or chaired multiple NIH study sections and editorial boards.
Abstract:
The HIV envelope protein (Env) is the sole virus protein expressed on the surface of virions and infected cells. Consequently, the development of anti-Env antibodies (Abs) for therapeutic applications is the subject of intense investigation. Anti-Env Abs can be used to neutralize cell-free virus and kill HIV-infected cells. After almost two decades with little progress, the introduction of recombinant DNA techniques has lead to a spate of highly effective and broadly reactive neutralizing Abs in the past ten years. HIV Env consists of two, non-covalently linked glycoproteins, the transmembrane anchor gp41, and the receptor-binding surface protein gp120. Neutralizing sites have been identified on both gp120 and gp41. Working with anti-HIV immunotoxins, we have also mapped the targets of these cytoxic agents. Interestingly, there was little correlation between neutralization and cell killing activities. In an effort to increase cytotoxic activity, we have made double variable domain (DVD) Abs that bind to structures on gp120 and gp41 that are the most effective targets for anti-HIV immunotoxins. Neither Ab, alone or in combination, neutralized HIV well. We were initially disappointed that the DVD’s offered no improvement in cytotoxicity over a mixture of the two Abs, but then bemused to discover that the DVDs were highly effective at neutralizing HIV infection. Activity was both broad and potent. I will discuss mechanisms whereby two weakly neutralizing Abs become more potent when combined into a single molecule.
Elizabeth J Goldsmith
UT Southwestern Medical Center at Dallas, USA
Title: The excursion model for MAP kinase signaling in health and disease
Time : 16:10-16:30
Biography:
Elizabeth J Goldsmith received her Ph. D. in Pysical Chemistry at UCLA, worked with Dave Eisenberg, Max Perutz, and Robert Fletterick, and has been at UT Southwestern for 27 years, in the Dept of Biophyscs. Her publications concern structural biology and biochemistry of protein kinases and other conformationally regulated proteins.
Abstract:
MAP kinase modules, a cascade of three kinase enzymes, a MAP3K, MAP2K and MAPK, propagate diverse extracellular signals to downstream effectors. The two double phosphorylation reactions catalyzed by the modules occur in a precise order that maximizes the number of reaction steps between signal and final activation. Activation is associated with the final Thr phosphorylation of the MAPK. The observed order of phosphorylation events suggests an excursion from the Ser/Thr kinase activity of the MAP3K into Tyr kinase activity of the central dual specificity MAP2K and back to Ser/Thr kinase activity. The reaction order, and thus the tempo of MAPK activation changes in cancer mutants of the MAP2K.
Takeshi Kikuchi
Ritsumeikan University, Japan
Title: Sequence analysis on the information of folding nuclei in proteins with interesting 3D properties
Time : 16:30-16:50
Biography:
Kikuchi has completed his PhD from Osaka City University and postdoctoral studies from Cornell University. Now, he is a professor of Department of Bioinformatics in Ritsumeikan University. Currently, he is interested in developing a new technique to predict 3D structural properties of a protein.
Abstract:
Extracting 3D structural information from a protein amino acid sequence is one of the significant problems in molecular bioinformatics. In particular, information on protein folding mechanism must be able to be decoded from the sequence of a protein if all information of the 3D structure is included in it. However, extracting the information on protein folding from its sequence is very difficult and the only limited information can be obtained by standard bioinfomatics techniques such as multiple sequence alignment or even more sophisticated techniques. Extracting 3D structural information from a protein amino acid sequence is one of the significant problems in molecular bioinformatics. In particular, information on protein folding mechanism must be able to be decoded from the sequence of a protein if all information of the 3D structure is included in it. However, extracting the information on protein folding from its sequence is very difficult and the only limited information can be obtained by standard bioinfomatics techniques such as multiple sequence alignment or even more sophisticated techniques. Our recent studies have demonstrated that the analyses of protein sequences based on the inter-residue average distance statistics with standard bioinformatics techniques and evolutionary analyses provide the information on protein folding nuclei. In the present study, we try to show the analyses of sequences of several protein families with specific 3D properties. We focus on the following properties; (1) similar partial 3D structures beyond a super family, (2) folding mechanisms of a 3D structure ubiquitously exists, (3) folding mechanisms of proteins with very highly homologous sequences but different 3D structures. In the present work, we take (1) E-to-H helix unit in globin family, (2) beta-Trefoil protein family, and (3) immunoglobulin binding proteins related proteins. We use the average distance map method and F value analysis which predict the compact regions and highly contact residues along the sequence of a given protein. Such information provides significant parts for protein folding along the sequence and also we compare such segments with the conservative hydrophobic residues and then we try to deduce the folding mechanism of a protein. In this way, we analyze the proteins in the categories above. We will present the possible folding mechanisms of these proteins based on the present analyses.
Barry K Hurlburt
United States Department of Agriculture, USA
Title: Ligand binding preferences of pathogenesis-related class 10 (pr-10) allergens
Time : 16:50-17:10
Biography:
Barry Hurlburt received his PhD in biochemistry from the University of Virginia and postdoctoral studies at Stanford University. In 1990 he became Assistant Professor at the University of Arkansas for Medical Sciences. He was promoted and tenured in 1997. In 2001 he moved to the USDA in New Orleans and is focused on peanut and tree nut allergies. He has published more than 50 papers in international journals and has recieved numerous extramural grants to support his research.
Abstract:
Rationale: Many PR-10 proteins are allergens when inhaled or ingested. One proposed function of these proteins is delivering bio-active compounds to wounds and/or the developing plant. We examined ligand binding to seven known PR-10 allergens. Ligand binding could well affect IgE binding. Methods: We generated pure, recombinant Ara h 8.01, Ara h 8.02, Cor a 1.02, Cor a 1.04, Que a 1.02, Que a 1.03 and Bet v 1.01 from peanut, hazelnut, white oak and birch respectively. Twenty three putative ligands were tested for binding using a fluorescence assay. Results: All of the proteins bound apigenin, daidzein, genistein, quercetin and resveratrol. Que a 1.03 bound the widest array of ligands including several fatty acids. Preliminary structural studies show changes in protein structure with ligand binding. Conclusions: Our results support the theory that these PR-10 allergens’ function in vivo is as a delivery vehicle for bio-active compounds. Now that we have identified biologically-relevant ligands we will test the possibility that binding them to PR-10 proteins may influence allergenic potential.
Olga I Podgornaya
Far Eastern Federal University, Russia
Title: Genomes repetitive sequences in progressive evolution
Time : 17:10-17:30
Biography:
Olga I Podgornaya is the Head of the Group of Non-coding DNA in the Institute of Cytology (INC RAS), professor at StPetersburg State University (SPbSU) and FEFU, Vladivostok. She was sent after graduation from SPbSU to the newly developed Far East RAS Center in Vladivostok. In 10 years she became a staff member in INC RAS. There she turned to gene engineering and DNA-binding proteins. When the iron curtain fell, Dr.Podgornaya worked in USA and United Kingdom, visited Canada, Germany, France. Grants from DOE USA (1993-2005) and Wellcome Trust (UK, 1992-1996) helped to survive during dark time for Russian science. She leads of the group of young researchers interested in eucaryotic chromatin structure and bioinformatics.
Abstract:
Transposable elements (TE) constitute no less than 48% of the human assembled genome and their positions determined. Alu repeats (SINE) are located mostly to the gene-rich regions, while LINEs enrich facultative heterochromatin. TE positions in both mouse and human genomes are very similar and cause the synteny. The comparison of the transcription intensity is done by bioinformatics tools in 32 human and mouse homologous tissues for ~12000 orthologous genes using transcriptome databases. Human shows a greater fraction of tissue-specific genes and a greater ratio of the total expression of tissue-specific genes to housekeeping genes in each tissue studied, which suggests a generally higher level of evolutionary cell differentiation (specialization). It could be supposed that TE positions fixed for ~65Myr (rodent and primate separation) and SINEs and LINEs attraction to the potentially activeinactive parts of the genome may be responsible for the chromatin landscape shaping. The substitution of four mouse SINEs with Alu-repeat correlate with the enhanced transcription. The large regions of classic heterochromatin enriched with tandem repeats (TR) poorly investigated though it is the mostly variable part of the genome. The TR or TE substitution in the mammalian genome could go on through sperm-mediated gene transfer followed by homologous recombination. The scheme suggested may remove two of the contradictions of the Modern evolutionary synthetic theory: (1) gene mutations are not necessary for the progressive evolution but repetitive elements substitution; (2) ancient mammalians could have litters with the number of offspring altered and inbred mating was possible for the descendant altered.
- Special Session on Prediction of protein-protein interaction in the Twilight Zone
Chair
Thomas Scior
Universidad Autonoma de Puebla, Mexico
Session Introduction
Thomas Scior & Angel Islas
Universidad Autónoma de Puebla, Mexico
Title: Prediction of protein-protein interaction in the twilight zone of low homology for the modulation of an ion channel: An analogy approach
Time : 17:30-18:00
Biography:
Thomas (alias Tom) Scior received his PhD as a German Pharmacist at the University of Tubingen (1994), did his postdoctoral training at CNRS, Strasbourg, France and became a professor and pioneered the computational molecular laboratory at the Pharmacy Department at Free Public University Benemérita Universidad Autónoma de Puebla, Mexico, (www.buap.mx); E-mail: tscior@gmail.com, thomas.scior @correo.buap.mx (deprecated), language skills (7), research skills (Computational Medicinal chemistry, computational molecular simulations by docking, SAR, QSAR, VS, MD, QM, bioinformatics, very low homology protein structure modeling and analogy models). CV METRICS > 80 undergraduate pharmacy courses; > 10 graduate students; 48 scientific publications; > 1000 scientific citations w/o auto-citations; so far predictive models were confirmed experimentally in one trial – one hit cycles: side directed mutagenesis and antidote against tetanus or botulinum for all vertebrates under international patent application (EP15186045.9, 2015). Memberships: ACS, AFM, AOG (Apotheker Ohne Grenzen), DPhG.
Abstract:
The tenet of homology modeling relies on the tendency that the divergence of structure directly reflects the variation in amino acid sequence identities. Hence the higher the sequence identity (homology) between a target and a template structure the more reliable the predicted target structure should be. This has been a useful paradigm to generate motifs, folds and domains of the proteins that have not been crystallized. Albeit, the study of dynamic protein-protein interactions (PPIs) that underpins natural phenomena – gating ions through cell membrane channels in the present case – requires the identification of functional interfaces. Here we demonstrate how a hitherto unknown PPI can be studied even under extremely low homology conditions (far distant phylogenic relationships) by an analogy protein modeling. This approach can overcome the problem of structural uncertainties due to random sequence similarities and can lead to additional insights where three-dimensional data is missing. It proposes – what we call – a common epi-homology feature which is neither structure-bound nor related to protein activity. Eventually, it was the distinction between reversibility and irreversibility of PPIs. The finding enabled us to identify two adjacent amino acids at the postulated reversible interface between two subunits (alpha and beta-1) of the skeletal muscle voltage-gated Na+ channel (Nav1.4). A single side-directed mutagenesis study combined with subsequent electrophysiological characterizations provided the proof of concept and validation. The outcome was a double mutant (T109A, N110A, called TANA for short) that caused the highest loss-of-function effect in the literature (“Identification of Nav β1 residues involved in the modulation of the sodium channel Nav1.4”; Islas AA, Sánchez-Solano A, Scior T, Millan-PerezPeña L, Salinas-Stefanon EM; PLoS One. 8(12): e81995; 2013; “Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1)”; Scior T, Paiz-Candia B, Islas ÁA, Sánchez-Solano A, Millan-Perez Peña L, Mancilla-Simbro C, Salinas-Stefanon EM; Comput Struct Biotechnol J. 13: 229-240; 2015).
Angel A Islas
Universidad Autónoma de Puebla, Puebla, Mexico
Title: Prediction of protein-protein interaction in the twilight zone of low homology for the modulation of an ion channel: An analogy approach
Biography:
Angel A. Islas received a BSc in Biology from the University of Puebla (BUAP), Mexico in 2007, a MRes in Medical and Molecular Biosciences with strand in Neurosciences awarded by the University of Newcastle Upon Tyne, United Kingdom in 2009 and a PhD in Physiological Sciences from BUAP in 2013. In 2014-2015, he carried out postdoctoral studies on Computational Biophysics at King’s College London in the United Kingdom. His postgraduate studies have been funded by the Mexican Council of Science and Technology (CONACyT). He was appointed research advisor in 2015 at the BUAP in Puebla, Mexico where he is currently collaborating with the department of Pharmacy and the laboratory of Biophysics at the Institute of Physiology. By combining electrophysiology with computational methods, his published work has contributed to the understanding of the modulation of the Na+ channel and the mechanism of action of K+ and Na+ channel inhibitors.
Abstract:
The tenet of homology modeling relies on the tendency that the divergence of structure directly reflects the variation in amino acid sequence identities. Hence the higher the sequence identity (homology) between a target and a template structure the more reliable the predicted target structure should be. This has been a useful paradigm to generate motifs, folds and domains of the proteins that have not been crystallized. Albeit, the study of dynamic protein-protein interactions (PPIs) that underpins natural phenomena – gating ions through cell membrane channels in the present case – requires the identification of functional interfaces. Here we demonstrate how a hitherto unknown PPI can be studied even under extremely low homology conditions (far distant phylogenic relationships) by an analogy protein modeling. This approach can overcome the problem of structural uncertainties due to random sequence similarities and can lead to additional insights where three-dimensional data is missing. It proposes – what we call – a common epi-homology feature which is neither structure-bound nor related to protein activity. Eventually, it was the distinction between reversibility and irreversibility of PPIs. The finding enabled us to identify two adjacent amino acids at the postulated reversible interface between two subunits (alpha and beta-1) of the skeletal muscle voltage-gated Na+ channel (Nav1.4). A single side-directed mutagenesis study combined with subsequent electrophysiological characterizations provided the proof of concept and validation. The outcome was a double mutant (T109A, N110A, called TANA for short) that caused the highest loss-of-function effect in the literature (“Identification of Nav β1 residues involved in the modulation of the sodium channel Nav1.4”; Islas AA, Sánchez-Solano A, Scior T, Millan-PerezPeña L, Salinas-Stefanon EM; PLoS One. 8(12): e81995; 2013; “Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1)”; Scior T, Paiz-Candia B, Islas ÁA, Sánchez-Solano A, Millan-Perez Peña L, Mancilla-Simbro C, Salinas-Stefanon EM; Comput Struct Biotechnol J. 13: 229-240; 2015).
Eduardo M Salinas-Stefanon
Universidad Autónoma de Puebla, Puebla, Mexico
Title: Prediction of protein-protein interaction in the twilight zone of low homology for the modulation of an ion channel: An analogy approach
Biography:
Eduardo M Salinas-Stefanon received his MD from the University of Puebla (BUAP), Mexico in 1980, he received his PhD in Physiological Sciences in 1996 awarded by University of Colima, Mexico. He carried out his postdoctoral studies under the supervision of Dr W. Giles at the University of Calgary, Canada in Pharmacology and Electrophysiology and with Dr P.H. Backx at the Centre for Cardiovascular Research, University of Toronto, Canada. He became a Full Professor in Physiology in 1984 at the Medical Faculty (BUAP), Mexico. He co-founded the Institute of Physiology BUAP in 1995, becoming head of the Department of Cardiac Biophysics. He became a member of the National Research System in 1993. His main research field includes cardiac physiology (pharmacology and biophysics) and protein-protein interactions of the subunits of the Na+ channel, including molecular dynamics of cardiac drugs. He is member of the Biophysical Society and SOBLA (Latin American Biophysical Society) since the 90s.
Abstract:
The tenet of homology modeling relies on the tendency that the divergence of structure directly reflects the variation in amino acid sequence identities. Hence the higher the sequence identity (homology) between a target and a template structure the more reliable the predicted target structure should be. This has been a useful paradigm to generate motifs, folds and domains of the proteins that have not been crystallized. Albeit, the study of dynamic protein-protein interactions (PPIs) that underpins natural phenomena – gating ions through cell membrane channels in the present case – requires the identification of functional interfaces. Here we demonstrate how a hitherto unknown PPI can be studied even under extremely low homology conditions (far distant phylogenic relationships) by an analogy protein modeling. This approach can overcome the problem of structural uncertainties due to random sequence similarities and can lead to additional insights where three-dimensional data is missing. It proposes – what we call – a common epi-homology feature which is neither structure-bound nor related to protein activity. Eventually, it was the distinction between reversibility and irreversibility of PPIs. The finding enabled us to identify two adjacent amino acids at the postulated reversible interface between two subunits (alpha and beta-1) of the skeletal muscle voltage-gated Na+ channel (Nav1.4). A single side-directed mutagenesis study combined with subsequent electrophysiological characterizations provided the proof of concept and validation. The outcome was a double mutant (T109A, N110A, called TANA for short) that caused the highest loss-of-function effect in the literature (“Identification of Nav β1 residues involved in the modulation of the sodium channel Nav1.4”; Islas AA, Sánchez-Solano A, Scior T, Millan-PerezPeña L, Salinas-Stefanon EM; PLoS One. 8(12): e81995; 2013; “Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1)”; Scior T, Paiz-Candia B, Islas ÁA, Sánchez-Solano A, Millan-Perez Peña L, Mancilla-Simbro C, Salinas-Stefanon EM; Comput Struct Biotechnol J. 13: 229-240; 2015).