Day 1 :
Henry M. Sobell completed his studies at Brooklyn Technical High School (1948-1952), Columbia College (1952-1956), and the University of Virginia School of Medicine (1956-1960). Instead of practicing clinical medicine, he then went to the Massachusetts Institute of Technology (MIT) to join Professor Alexander Rich in the Department of Biology (1960-1965), where, as a Helen Hay Whitney Postdoctoral Fellow, he learned the technique of single crystal X-ray analysis. He then joined the Chemistry Department at the University of Rochester, having been subsequently jointly appointed to both the Chemistry and Molecular Biophysics departments (the latter at the University of Rochester School of Medicine and Dentistry), becoming a full tenured Professor in both departments (1965-1993). He is now retired and living in the Adirondacks in New York, USA.
Premeltons are examples of emergent structures (i.e., structural solitons) that arise spontaneously in DNA due to the presence of nonlinear excitations in its structure. They are of two kinds: B-B (or A-A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally nontopological, undergo breather motions that allow drugs and dyes to intercalate into DNA. B-A (or A-B) premeltons, on the other hand, are mobile, and being globally topological, act as phase-boundaries transforming B- into A- DNA during the structural phase-transition. They are not expected to undergo breather-motions. A key feature of both types of premeltons is the presence of an intermediate structural-form in their central regions (proposed as being a transition-state intermediate in DNA-melting and in the B- to A- transition), which differs from either A- or B- DNA. Called beta-DNA, this is both metastable and hyperflexible – and contains an alternating sugar-puckering pattern along the polymer-backbone combined with the partial-unstacking (in its lower energy-forms) of every other base-pair. Beta-DNA is connected to either B- or to A- DNA on either side by boundaries possessing a gradation of nonlinear structural-change, these being called the kink and the antikink regions. The presence of premeltons in DNA leads to a unifying theory to understand much of DNA physical-chemistry and molecular-biology. In particular, premeltons are predicted to define the 5’ and 3’ ends of genes in naked-DNA and DNA in active-chromatin, this having important implications for understanding physical aspects of the initiation, elongation and termination of RNA-synthesis during transcription. For these and other reasons, the model will be of broader interest to the general audience working in these areas. The model explains a wide variety of data, and carries within it a number of experimental predictions – all readily testable – as will be described in my talk.
- Sobell HM, (2016) Premeltons in DNA. Journal of Structural and Functional Genomics 17:17-31.
- Sobell HM, (2013) Organization of DNA in Chromatin. Rather than bending uniformly along its length, nucleosomal DNA is proposed to consist of multiple segments of B- and A- DNA held together by kinks when forming its left-handed toroidal superhelical structure. Explanatory publications. ISBN 978-0-692-01974-0.
RIKEN Structural Biology Laboratory
Shigeyuki Yokoyama obtained his PhD degree from The University of Tokyo in 1981. He was associate professor (1986–1991) and professor (1991–2012) at The University of Tokyo, and now is emeritus professor. He was also appointed 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 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.
The adiponectin receptors, AdipoR1 and AdipoR2, are key anti-diabetic molecules. AdipoR1 and AdipoR2 are seven transmembrane helix receptor proteins orienting their N- and C-termini on the intracellular and extracellular sides, respectively, which is opposite to G-protein coupled receptors (GPCRs). We determined the crystal structures of human AdipoR1 and AdipoR2, and found that they represent a novel class of receptor structure. The seven transmembrane helices form a large internal cavity, in which three conserved His residues coordinate a zinc ion. This zinc-coordinated structure indicates that AdipoR1 and AdipoR2 are hydrolytic enzymes. Both AdipoR1 and AdipoR2 assume the closed and open forms. The lipids bound in the closed and open forms were identified, which indicated that the zinc-coordinated structure is for lipid hydrolysis.
We determined the crystal structure of a GPCR, leukotriene B4 (LTB4) receptor BLT1, bound with an antagonist. BLT1 exhibits the canonical seven transmembrane helix structure. The binding mode of the antagonist is characteristic, and is expected to be useful for further drug development.
We applied the cell-free protein synthesis method to production of GPCRs. By adding a mixture of mammalian lipids in the cell-free reaction, GPCRs were synthesized and folded with lipids. This method is useful for large-scale production of high quality GPCR samples for structural and functional studies.
- Muramatsu T, Takemoto C, Kim YT, Wang H, Nishii W, Terada T, Shirouzu M, Yokoyama S (2016) SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity. Proc. Natl. Acad. Sci. 113(46):12997-13002.
- Shinoda T, Shinya N, Ito K, Ohsawa N, Terada T, Hirata K et. al. (2016) Structural basis for disruption of claudin assembly in tight junctions by an enterotoxin. Sci. Rep. 6:33632.
- Shinoda T, Shinya N, Ito K, Ishizuka-Katsura Y, Ohsawa N et. al. (2016) Cell-free methods to produce structurally intact mammalian membrane proteins. Sci. Rep. 6.
- Kashiwagi K, Takahashi M, Nishimoto M, Hiyama TB, Higo T et. al. (2016) Crystal structure of eukaryotic translation initiation factor 2B. Nature. 531(7592):122-125.
5. Tanabe H, Fujii Y, Okada-Iwabu M, Iwabu M, Nakamura Y et. al. (2015) Crystal structures of the human adiponectin receptors. Nature. 520(7547): 312-316.
University of Basel, Switzerland
Tilman Schirmer is interested in molecular mechanisms of bacterial signal transduction. He is a trained Crystallographer, but has extended his activities into functional characterization and kinetic modeling to reveal structure - function relationships. He has graduated at the Max-Planck Institute in Martinsried (Germany) and has worked at the LMB Cambridge (UK) on the regulation of phosphofructokinase. He then moved to the Biozentrum Basel (Switzerland) to reveal the structure and translocation mechanism of maltoporin. His current interest lies mainly in the various aspects of c-di-GMP signaling and FIC-mediated AMPylation of target proteins.
In addition to the well-known cyclic nucleotides, cAMP and cGMP, bacteria utilize cyclic di-guanosine monophosphate (c-di-GMP) to control various cellular processes. Hereby, the cellular level of the messenger is set by the antagonistic activities of diguanylate cyclases and specific phosphodiesterases. In a given organism, there are usually multiple variants of the two enzymes, which are tightly regulated by a variety of external and internal cues due to the presence of specialized sensory or regulatory domains. Fundamental cellular processes, such as bacterial life style, biofilm formation, and cell cycle control are thus getting controlled in a coordinated fashion by downstream c-di-GMP receptors in response to the input signals.
Crystal structures in combination with biochemical and biophysical analyses reveal that both GGDEF diguanylate cyclase and EAL phosphodiesterase domains are active only as homotypic dimers. In the full-length enzymes, attainment of the competent quarternary structure depends on the signaling state of the accessory domains (e.g. Rec, PAS, GAF), that typically dimerize or change their dimeric structure upon signal perception. Histidine kinases and transcription factors use very similar regulatory domains to control output function in a dimeric context. It can be inferred that the modular arrangement of catalytic and regulatory dimers, both forming homotypic interactions, facilitates their recombination during evolution.
As an example for c-di-GMP mediated allosteric control of a downstream effector, the effect of c-di-GMP binding to the bifunctional histidine kinase CckA from C. crescentus will be presented. It was found that c-di-GMP promotes the phosphatase activity of the enzyme via stabilization of the phosphatase competent constellation due to non-covalent domain cross-linking. In silico analyses predict that c-di-GMP control is widespread among bacterial histidine kinases, arguing that it can replace or modulate canonical transmembrane signaling.
- Schirmer, T (2016) C-di-GMP Synthesis: Structural Aspects of Evolution, Catalysis and Regulation. J. Mol. Biol. 428(19):3683–3701.
- Dubey B, Lori C, Ozaki S, Fucile G, Plaza Menacho I, Jenal U, and Schirmer T (2016) Cyclic di-GMP mediates a histidine kinase/phosphatase switch by noncovalent domain cross-linking. Science Advances. 2(9):e1600823.
- Reinders A, Hee C S, Ozaki S, Mazur A, Boehm A, Schirmer T, Jenal U (2016) Expression and Genetic Activation of Cyclic di-GMP-specific phosphodiesterases in escherichia coli. Journal of Bacteriology. 198(3):448–462.
- Sundriyal A, Massa C, Samoray D, Zehender F, Sharpe T, Jenal U, Schirmer T (2014) Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger cyclic di-GMP. J. Biol. Chem. 289(10):6978–6990.
- Schirmer T, Jenal U (2009) Structural and mechanistic determinants of c-di-GMP signalling. Nat. Rev. Microbiol. 7(10):724–735.