Day 2 :
University of Georgia, USA
Keynote: The use of soft x-ray diffraction for native-sad and for chromatic exploration of uncharted aspects of metals in macromolecules: A new pilot program at the aps
Time : 09:00-09:40
Bi-Cheng (B.C.) Wang completed his PhD from the University of Arkansas and postdoctoral studies from the California Institute of Technology. He currently holds the title of Professor and Ramsey Georgia Research Alliance Eminent Scholar at the University of Georgia. He is the founding Director of SER-CAT (Southeast Regional Collaborative Access Team, www.ser-cat.org), which includes the construction and operations of two synchrotron beamlines at the Advanced Photon Source, USA. He has also served as the PI and Program Director of the NIH-funded Southeast Collaboratory for Structural Genomics (SECSG). He has published more than 200 papers in peer-reviewed journals.
Native-SAD phasing uses the anomalous scattering signal of atoms in the crystalline, native samples of macromolecules, collected from single-wavelength X-ray diffraction experiments. Advances in technology and methodology during the past five years show great promise in making Native-SAD phasing a routine approach (Rose, Wang & Weiss, 2015) for future macromolecular structure determination using X-rays with wavelengths longer than 1.5 Å. Another use of the X-rays in this wavelength region is to collect multiple sets of diffraction data across a metal’s absorption edge as one does in X-ray Absorption Spectroscopy (XAS). The diffraction-based approach differs from XAS in that a complete 3-dimensional diffraction data set is recorded at several points spanning the metal’s absorption edge. The approach provides both positional and spectroscopic information relating to individual metals in the crystal by diffraction. A Pilot Program was initiated at the APS in January 2016 for General Users to participate in the use of these soft X-ray tools. Theoretical and practical aspects of the above concepts, as well as the procedures for accessing the new Pilot Program through General User beam time, will be introduced.
University of California, San Francisco, USA
Time : 09:40-10:00
Robert M. Stroud is a Professor of Biochemistry and Biophysics at UCSF. He obtained his BA, MA at Cambridge U niversity, and his PhD in J.D. Bernal’s laboratory at London University where he programmed non-centrosymmetric direct methods to determine structure of the nucleoside antibiotic tubercidin, and vitamins. He was postdoctoral with R. E. Dickerson where he determined the first structure of trypsin and trypsinogen . He became associate professor of chemistry at Caltech, and moved to UCSF as the founding memb er of the Macromolecular Structure Group (msg.ucsf.edu). He is a member of the National Academy of Sciences USA, a Fellow of the American Academy of Arts and Sciences, and a Fellow of the Royal Society of Medicine (UK). Stroud’s rese arch focuses on the mechanisms of transmembrane transporters, receptors and cha nnels, and on the mechanisms of protein-RNA recognition and chemistry, at the level of atomic structure , mechanisms, inhibition, and structure-based drug discovery.
The structure determination of an integral membrane protein complex of an ion regulated, voltage gated two-pore channel TPC-1 is reported at 2.9Å resolution. The channel is gated by external Ca++ ions and by voltage such that at zero voltage the channel is closed, in its resting state. In other voltage gated channels the sensor is activated leading to a comparison of the two states that provides the first example of what kinds of transitions occur in an inactive-to-active state in a voltage sensitive channel. This leads to a proposal for the mechanism of voltage sensors that control channel activity. In addition the channel controls an intracellular organelle that controls viral entry into cells for membrane encapsulated filoviruses. An inhibitor NED19 is bound that shows how the channel is inhibited allosterically, at a distance from the channel. Inhibition of TPC1 by NED19 leads to a cure for Ebola infected mice.
- Special Session on Modularity and metastability: Structural aspects of nanomedicine
R Hollang Cheng
University of California, Davis, USA
High Energy Acclerator Research Organization (KEK), Japan
University of California, Davis, USA
Time : 10:20-10:40
R Holland Cheng, Ph D is a Professor of Molecular and Cellular Biology at University of California. Cheng received a master degree in 1989 and a Ph D degree in 1992 from Purdue University. Cheng has served as a Panel Reviewer for NIH programmed project grant, NIH Microbiology & Infectious Diseases Research Committee, NIH National Center for Research Resources, Wellcome Trust, UK and Earth & Life Sciences Council, Netherlands (2002). Cheng has actively served on the advisory board of the International Microscopy Congress plus regional microscopy societies, American Biographical Institute, and International Virus Assembly Symposium.He is also a regular member of the American Chemical Society,American Society for Microbiology and American Biophysical Society . Cheng has published nearly fifty papers in peer reviewed journals and his research mainly focuses on Proteome imaging of macromolecular systems.
High Energy Accelerator Research Organization (KEK), Japan
Time : 10:40-11:00
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).
Helicobacter pylori , a gram-negative bacterium that colonizes the human gastric mucosa , is recognized as a major risk factor for gastric diseases, such as peptic ulcers and gastric carcinomas . H. pylori delivers an effector protein CagA into gastric epit helial cells and th e EPIYA and CM segments in the C-terminal intrinsically disordered (ID) region of CagA (CagA-C) promiscuously interact with cellular signaling molecules, such as SHP2 and PAR1b, to deregulate these signaling proteins. To analyze the structure and function of a large protein complex formed by CagA, SHP2, and PAR1b, we started a structural biology study of the CagA-SHP2-PAR1b complex. Since our group already determined the crystal structure of the N-terminal structured region of CagA (CagA-N) (1), we are working on structure and function of CagA-C. Ou r biochemical analysis suggested that the CBS segment in the CagA-C region interacts with the NBS segment in CagA-N, forming a four-α-helix bundle structure and the CagA-C region adops a lariat-loop like structure. Interestingly, mutations in the NBS and CBS segments caused a reduced biological activity of CagA (humminbird-inducing ability in AGS cells) (1). For further understanding of the structure-function relationship of CagA-C, we have confirmed the existence of the lariat-loop like structure. In addition, we have obtained some structural information of the CagA-C region and its interaction with SHP2. We will discuss the structural characteristics of CagA-C and its relationship to the biological activities.
University of Tampere, Finland
Time : 11:20-11:40
Vesa P Hytonen is a head of the Protein Dynamics research group at the University of Tampere. After graduating as a PhD from the University of Jyväskylä, Jyväskylä, Finland at 2005, he conducted postdoctoral training at ETH Zurich, Zürich, Switzerland 2005-2007. He then continued as a postdoctoral researcher at the University of Tampere and established independent res earch group at 2010. His research interests are mechanobiology , protein engineering and vaccine research, and he has authored more than 90 scientific articles.
Talin is cytoplasmic protein essential for cell adhesion. It connects integrin receptors for actin cyto skeleton and acts as a mechanosensor by recruiting vinculin molecules as a response to mechanical stress applied on it. We have studied tal in structure and function using extensive molecular dynamics simulations , small-angle X-ray scattering , cryo-EM and biophysical methods combi ned with cell biology methods. Th ese studies have revealed novel insights into role of talin in integrin activation. We have also been able to develop more detailed picture of about the role of talin rod in mechanosensing and in regulation of cell adhesion dynamics, cell migration and traction force generation.
Lawrence Berkeley National Laboratory, USA
Title: Individual-Particle Electron Tomography (IPET): an approach to study flexible protein structure and dynamics
Time : 11:40-12:00
Ren is a staff scientist and lab director at Molecular Foundry, LBNL. He received his BS and MS in theoretical physics at Lanzhou University and PhD in material physics at Univ of Science and Tech Beijing. He received his postdoc training at Scripps Research Institute. He started his r esearch lab at UCSF in 2006 and then transfer to LBL in 2010. His research group is supported by DOE, NIH and Instrudy funds. He published more than 70 papers in reputed journals and has been serving as an editorial board member of numerial journals.
Proteins have the unique ability to function specifically and efficiently, which is attained through its three-dimensional (3D) structures and flexibility, as well as necessary conformational changes. However, structural study on proteins that have large-scale flexibility, dynamics, and heterogeneity is challenging by current techniques, including X-ray crystallography , nuclear magnetic resonance (NMR) spectrum, small angle scattering (SAXS) and electron microsc opy (EM) single-particle reconstruction. A fundamental approach to study the structure of flexible proteins should be based on the signal from each individual protein molecule itself instead of averaging fr om different protein molecules. EM provide a novel tool to image each individual molecule at atomic resolution level; while electron tomography (ET) provide an approach to image a targeted molecule from a series of tilt angles. Although the signal obtained from an individual molecule has been believed for decades to be too weak to achieve any 3D structure with a meaningful resolution, we recently re-investigated this possibility carefully and proposed an individual-particle electron tomography ( IPET) approach with a ‘‘focused electron tomography reconstruction’’ (FETR) algorithm to improve the 3D structure resolution via decreasing the reconstructing image size with an iterative refinement process. IPET does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can provide near one nanometer resolution 3D structure from an individual protein molecule. Through the structure determination of each individual molecule, the compar ison of these molecular structures provides a new opportunity to reveal the dynamic character, equilibrium fluctuation, mechanism, aggregation and even structural changes in proteins during a chemical reaction or biological event.
Hamad bin Khalifa University, Qatar
Title: Integrated methods enable re-engineering of transcription factors allowing cell fate modification
Time : 12:00-12:20
Prasanna Kolatkar completed his PhD at the University of Texas at Austin in 1991 in the Department Chemistry and Biochemistry and post-doctoral studies in the laboratory of Michael Rossmann at Purdue University where he received a Ja ne Coffin Childs Memorial Fund fellowship. Upon moving to Singapore in 1997, he worked at the Bioinformatics Center and Institute of Molecular and Cell Biology in Singapore before joining the Genome Institute of Singapore in 2001. Subsequently he jo ined QBRI in 2013. Recently his work has focused on re-engineering of stem cell function through directed mutagenesis of key residues involved in protein-protein interactions.
Transcription factors (TFs) are key players in early cell development which direct cells towards specific fates. The Sox family of TFs is composed of 20 members which are responsible for directing a wide variety of fates. The ability of highly similar TFs to carry out such distinct outcomes had been a conundrum to date. We have studied several Sox-DNA (HMG domain) structu res in my laboratory which all display essentially similar structures. The Sox binding motif is only 6-8 bp long and this once again raises the question as to how specificity can be achieved. We discovered that a critical Sox17 residue involved in protein interaction with its partner Oct4 (POU domain) helps to install specificity of binding to its cognate DNA and consequently give rise to endoderm . Subsequently we were able to re-engineer Sox17 through a single resid ue mutant and convert it into a potent pluripotent TF by substituting Sox2 in the standar d induced pluripotent stem cell cocktail composed of 4 TFs. Next we also studied the genomic profiles of native and mutant Sox TFs through a battery of ChIP-seq experiments to validate the in-vitro results. The integrated application of structural and genomic methods have allowed us to analyze and understand how highly similar TFs can partner each other through key interaction points which give rise to highly specific outcomes. TF function can be altered through modified TF forms as well as novel ligands which could have applications in disease and therapy.
High Energy Accelerator Research Organization (KEK), Japan
Time : 12:20-12:40
Miki Senda has completed her PhD at 2008 from Nagaoka University of Technology. She is an assistant professor of Structural Biology Research Center in High Energy Accelerator Research Organization (KEK). She have several collaborations, in which she has worked as an expert of protein crystallization and crystal quality improvement. She received Oxford Cryosystems Low Temperature Prize at the 63rd Annual meeting of the American Crystallographic Association (ACA) in 2013.
Current X-ray sources of synchrotron radiation enable us to determi ne the crystal structures of proteins even if the crystals were diffracted only to medium or low resolution. However, high-resolution crystal structures ar e still required for the pharmaceutical and biochemical sciences . When obtained crystals were of poor quality and insufficient for crystal structure determination, post-crystallization treatment could improve the crystal quality. Our experiences of crystal structure analyses of hist one chaperon T AF-Iβ, CagA oncoprotein from Helico bacter pylori and GTP sensor PI5P4Kβ showed that crystal soaki ng into cryoprotectants improved crystal-quality (1, 2, 3). Of the three proteins, crystal qualities of CagA and PI5P4Kβ were significantly improved by using more than one cryo-protectant. This method, multi-step soaking method, improved not only the maximum resolution but also success rate of high resolution data collection. Reproducibility of the crystallization is another critical problem in determining crystal structure. Anaerobic crystallization and immediate observation method are effective to improve the reproducibility of the crystallization. We would like to report some examples of the crystallization and crystal quality improvement by our strategy.
University of Florida, USA
Title: Symmetry ambiguity and its implications to the molecular functions of the MAVS prion-like filaments
Time : 12:40-13:00
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.
The intracellular innate immune response takes advantage of the induced filament formation i n order to achieve high sensitivity and signal amplification . The RIG-I –MAVS signaling is initiated by the Rig-I sensing of individual viral RNA (vRNA) molecules, which induce the formation of RIG-I/vRNA dimers. The activated RIG-I dimers together with poly-ubiquitins trigger the self-inhibited MAVS molecules on the outer surface o f mitochondria to release their CARD domains, interact with each other and f orm fibril structures. We have been working on unde rstanding the structural basis for the RIG-I –MAVS signaling, both the vRNA-induced RIG-I dimerization and the switch of MAVS monomers from the self-inhibited state to the filament form both in vitro and in virus-infected cells. We observed significant heterogeneity in the MAVS filaments. Symmetry analysis of the filaments showed ambiguity in them. In our earlier datasets, a majority of our images agreed better with the C3 symmetry, which led us to prepare filaments under low ionic strength to increase electrostatic interaction and filament quality. Sorting of the cryoEM images from these filaments led to a significant portion of the data that showed C1 symmetry, consistent with a group of fil aments formed by renaturization of the denatured CARD domsignal amplification poly-ubiquitins fibril structures signaling cryoEM images CARD mutagenesisains. We performed parallel sorting of our dat asets using different algorithms against two different structural models to estimate the heterogeneous composition in the datasets. The two models are not well distinguished by the mutagenesis data we have collected. They suggest two different molecular mechanisms and need further experimental examination. A major open question is which one or both filaments are formed inside the virus-infected cells.
GlaxoSmithKline Vaccines S.r.l., Italy
Title: Crystal structure of a human Fab in complex with a dominant antigen from Neisseria meningitidis
Time : 13:40-14:00
Jacinto López-Sagaseta completed his PhD on 2007 and carried out a postdoctoral stage at the University of Chicago. His work has been focused mainly in the characterization of the endothelial protein C receptor and its interaction with the coagulation Factor VII. During his postdoctoral research, he pursued structural studies on antigen recognition by NKT and MAIT cells. He has also worked on the structural elucidation of a potassium ion channel bound t o an inhibitory ligand . In 2015, Jacinto López-Sagaseta was awarded a Marie Curie European Grant and joined the Structural Biology Unit of GSK Vaccines where he is carrying out studies in the context of structural vaccinology.
Anchored yet exposed to the outside moiety of the bacterial shell, Factor H binding protein (fHbp) is one of the main antigenic components of Neisseria meningitidis, one of the causative agents of men ingitis , an infect ious disease that can cause a fatal outcome or permanent disability within 24 hours of infection. Though there have been described up to three different variants of fHbp, it is fHbp variant 1 (fHbp-1), the subclass showing the highest prevalence amongst MenB strains, and also, one of the actual components of Bexsero, the current licensed vaccine against serogroup B Meningococci (MenB). In order to define the structural basis that underlie the recognition of this highly immunogenic antigen and the broad strain coverage offered by Bexsero, we have determined the crystal structure of a complex between a human Fab and fHbp-1 at a resolution of 2.2 Å. The Fab has been originated from an immunization study that included a recombinant form of fHbp-1, and importantly, it is cross-reactive against all of them. The cross-reactive epitope spans along the c-terminal beta barrel of fHbp and encompasses residues that are highly conserved across the different fHbp variants. The hypervariable CDR3 loop of the heavy chain dominates the recognition of the antigen. This crystal structure represents the first evidence, at the atomic level, of the recognition of Neisseria meningitidis fHbp by a human Fab r aised in an individual upon vaccination, and provides the basis behind the broad strain coverage of the current vaccine against MenB. In addition, the information gathered from this structure will be of high value for future structure-based antigen design.
National Sun Yat-sen University, Taiwan
Title: The atomic structures of dragon grouper nervous necrosis virus from cryo-EM in comparing with X-ray diffractions
Time : 14:00-14:20
Chan-Shing Lin has completed his PhD at University of California, Irvine, in 1992. He has published more than 65 papers in reputed journals and has been serving as an Editorial Board Member of Archive of Virology and Frontier journals. He has also consulted biotechnology companies in building the cGMP processes for manufacturing vaccines and bio-medicals.
Betanodaviruses cause lethal nervous necrosis in more than f orty species of fish. I will present the maps from cryo-electron microscopy (cryo-EM), since 2001 when the first map was published, in comparison to x-ray diffractions . During the low resolution era of 10-20 Å maps, the molecular features of the Dragon Grouper Nervous Necrosis Virus (DGNNV) were intensively studied. Virus-like particles (VLPs) are formed by the single capsid p rotein that was expressed either in E. coli, yeast, or insect cell, while VLPs were not found when 35 amino acids at the N-terminus or four amino acids at C-terminus were deleted. Residues of aspartic acids interacting with cations are found to be important to the VLP stability, whereas the potential disulfide bounds of cysteine residues were not essential for the particle assembly. In the pathway of the DGNNV entry into fish cells, micropinocytosis interacting with heat-shock protein HSP90-like was proposed while the detail interaction of protrusion domain with lipid-bound protein is under investigated using x-ray refractivity. Three domains of the capsid protein (RNA-binding, shell, and protrusion domains) in truncated clones were determined to 4-6 A using x-ray diffraction. The proposed structure of the Betanodvirus since 2001 was doubly confirmed, in addition to another cation-binding site at protrusion was found. Recently, cryoEM with a direct detection camera to overcome specimen motion and radiation damage issues provides near atomic structures of the T=3 DGNNV VLPs. The structure of the shell domain (52-213th aa) in weak basic condition was determined to 3.56 Å resolution and an atomic model that was built de novo reveals protein-protein interactions , calcium-ion bridges, and unique cation interactions. The cation interactions stabilize the particle by holding three subunits in an asymmetric unit of trimer and p atching asymmetric units into an icosahedron and their alterations have led to expansion or disruption of the particle. The cryo-EM structure changes upon immersion in an acidic condition that mimics endocytosis entry pathway suggests that a pH-sensing mechanism for the piscine NNV machinery to deliver its genome.
- Track 7: Molecular Modelling | Track 8: Complexity Arenas in Structural Biology | Track 9: Structural Biology in Cancer
Jagiellonian University, Poland
University of Texas Southwesten
Jagiellonian University – Medical College Krakow, Poland
Time : 14:20-14:40
Irena Roterman-Konieczna has completed his PhD at the age of 35 years from Nicolaus Copernicus Medical Academy Krakow, Poland - postdoctoral studies from Cornell University Ithaca NY USA – Harold Scheraga Group. She is the director of Department of Bioinformatics and Telemedicine at Jagiellonian University – Medical College. She has published more than 25 papers in reputed journals and has been serving as an editorial board member of repute. The field of interst is the protein structure and folding simulation as well as systems biology. General model for protein folding simulation is published by Woodhead Publishing (currently Elsevier). The model for proteome construction is presented in the book published by Springer. She is the Chief Editor of the journal Bio-Algorithms and Med-Systems (de Gruyter).
Fuzzy oil drop model - in contrast to discrete oil drop - introduces the gradual decrease of hydrophobicity from the center of protein molecule (the highest concentration of hydrophobicity) reaching zero level on the surface of protein body. The 3D Gauss function is assumed to represent the idealized hydrophobicity distribution in protein molecule. The real hydrophobicity distribution may differ since it is the result of residues distribution and the pair-wise hydrophobic interaction depending on the intrinsic hydrophobicity of each residue. The difference between idealized distribution and observed one can be measured quantitatively applying the Kullback-Leibler divergence entropy. It measures the distance between observed versus idealized hydrophobicity distribution in the protein as a whole and is able to identify the fragments of high and low similarity. In consequence the localization of high and low stability may be identified since hydrophobic core is assumed to be responsible for tertiary structure stabilization. The directing hydrophbic residues toward the center of protein is the effect of external force field (water environment) during folding process. The balance between external force field (water) and internal force field (non-bonding interaction between toms in protein) is assumed to be the mechanism of protein folding. Local disorder of hydrophobic core (versus idealized one) is quite often related to biological activity (ligand/substrate binding – local hydrophobicity deficiency, protein-protein complexation – local excess of hydrophobicity).
University of Texas Southwestern Medical Center at Dallas, USA
Time : 14:40-15:00
Jyh-Yeuan (Eric) Lee completed his PhD from the University of California, Riverside, in cryo-electron microscopy (cryo-EM) and drug resistance ABC transporters. He has performed his postdoctoral st udies in large-scale membrane protein expression and puri fication of human ABC transporters and X-ray crystallography of the sterol transporter ABCG5/ABCG8 from Texas Tech University Health Sceinces Cent er and the University of Texas Southwestern Medical Center at Dallas. He is currently a postdoctoral research scientist at the McDermott Center in Human Growth and Development at the University of Texas Southwestern Medical Center at Dallas.
ATP binding cassette (ABC) transporters play critical roles in maintaining stero l balance in higher eukaryotes , as exemplified by the ABCG5/ABCG8 heterodimer (G5G8) that mediates sterol excretion in liver and intestines. Mutations disrupting G5G8 cause sitosterolemia, a disorder characterized by sterol accumulatio n and premature atherosclerosis . Here we use crystallization in lipid bilayers to determine the X-ray structu re of huma n G5G8 i n a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC st erol transporter. The structure reveals a new transmembrane fold that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains (TMD) are couple d to the nucleotide-binding sites (NBS) by networks of interactions that differ between the active and inactive ATPases , reflecting the catalytic asymmetry of the transporter. We discovered the TMD polar network that may play a role in transmitting signals from ATPase catalysis in the NBS to sterol transport on the TMD. Based on molecular dynamic simulation and long-range coevolution analysis, as well as in vivo structure-based functional mutagenesis , we propose an updated model for sterol transport mechanism. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolemia, and will serve as a new structural template for homology modelling to a wide range of transport system that are regulated by ABCG transporters.
United States Department of Agriculture, USA
Time : 15:00-15:20
Maleki is an expert in food allergy research and has worked in this field for over 18 years. She recieved her Ph.D. from the Univ of Arkansas for Medical Sciences. She has served on the Scientific Advisory Committee to the FDA, the Nataional Peanut Board, and NIH’s Guidelines of the Diagnosis and Management of Food Allergy and as Expert Reviewer for the ($70 million) Canadian AllerGen project. She is a Fellow of the AAAAI and has edited a book and authored over 90 publications, and has had a significant number of invited national and international speaking and reviewer engagements.
Food allergy is on the rise and the prevalence of peanut allergy has more than tripled in the U.S. in the last 20 years. Mea nwhile, little is known about why certain proteins in foods are allergenic and others are not. Also, it is important to know what happens to the allergenicity of food products after pr ocessing. To assess processing-induced changes , the major allergen s were purified from raw (R), and roasted (Ro) peanuts and the structure and IgE binding were compared with various ELISA and immunoblots with allergic sera , circular dic hroism , mass spectroscopy and e nzymatic digestion. While the structure of the allergens purified following roasting did not show significant changes compared to the raw, the IgE binding and SPT to the roasted samples peanuts were higher. Although allergen structure was found to be more important than linear sequence, it is likely that the roasting-induced chemical modifications are more important for enhanced IgE binding and immunogenicity than the structural changes to the major peanut allergens. Mass spectroscopic analysis was utilized to identify specific processing-induced chemical modif ications of the peanut allergens that may contribute to the observed effects. Thermally processed peanut proteins are chemically modified, covalently cross-linked, less soluble, more resistant to digestive enzymes, bind higher levels of IgE, and cause higher skin prick test (SPT) reactivity than raw peanut proteins. This knowledge may be useful in the development of more specific and improved diagnostic, therapeutic and detection tools and potentially lead to development of processes that can result in reduced allergenicity of a food.
Academia Sinica, Taiwan
Title: Structural analysis of PTPN3-p38y interactions by chemical cross-linking coupled with mass spectrometry
Time : 15:20-15:40
Tzu-Ching has completed his PhD from University of Nebraska Medical Center in 1999 and postdoctoral studies from Cold Spring Harbor Laboratory in 2003. Since then, he has been working at Academia Sinica, the premier government-funded institution in Taiwan. He is now a Research Fellow with professorship jointly appointed by National Taiwan University. He has published more than 40 papers in reputed journals and has been serving as an advisory board member of competitive journals.
Purpose: Protein tyrosine phosphatase N3 (PTPN3) and mitogen-activated protein kinase p38 y coordinae to promote Ras-induced oncegenesis. Structural analysis of PTPN3-p38y complex is a challenging task by using traditional x-ray crystallography . Therefore, chemical cross-linking coupled with mass spectrometry (CX-MS) has been developed as an alternative method to solve this obstac le. Experimental description: Through covalent linkage of two spatially proximate residues within a single or between two polypeptide chains, the CX-MS approach provides structural insights into the flexible regions of proteins under any circumstances amenable to analysis. Convention ally, mapping of the protein-protein interaction sites relies on the coupling of lysine residues on the surface. Together with the distance restraint of the selected cross-linker, the segments of interaction surface can be determined. Results: Employing this method, we have mapped the contact interfaces of the PTPN3-p38y complex. Our finding of the solution structure indicated that the catalytic domain of PTPN3 interacts with the activation loop of phosphorylated p38y, illustrating how PTPN3-mediated dephosphorylation of p38y takes place. The CX-MS approach further demonstrated that the PDZ domain of PTPN3 recruits the PDZ-binding motif of p38y, thus stabilizing the active-state complex of PTPN3-p38y. Moreover, the CX-MS analysis defined the autoinhibitory characteristic of the PDZ domain in PTPN3 in the absence of p38y. Conclusions: By combining other approaches such as small-angle x-ray scattering (SAXA) and crystal structure of PTPN3-phospho-p38y peptide, the CX-MS method generates considerable insights into the architecture of the phosphatase-kinase complex assembly.
Ritsumeikan University, Japan
Title: Predicting the binding free energy of the inclusion process of Cyclodextrin deriveatives and small molecules by means of MM/3D-RISM method
Time : 16:00-16:20
Mar. 2014, Ph. D, Ritsumeikan University Apr. 2014 - Mar. 2015, Postdoctoral Fellow, Ritsumeikan University Apr. 2015 - present, Assistant Professor, Ritsumeikan University During the Ph. D course, I worked with Prof. Takeshi Kikuchi and tried to apply a course grained simulation for analyzing folding mechanism of proteins. Since 2014, I have been working with Prof. Fumio Hirata and trying to apply 3D-RISM theory to the drug screening and to develop new methods based on 3D-RISM theory.
Cyclodextrin is a cyclic molecule formed by s ix to eight glucopyranose units and i nclude a small hydrophobic molecule into a cavity. Cyclodextrins are widely used as a additive of foods and drugs , because cyclodextrins have low toxicity and its physicochemical characteristics can be varied by the substitution of hydroxyl group. Although rational design of cyclodextrin derivatives are desired to develop a new pharmaceutical products based on inclusion ability of the cycrodextrins, designing of a functional Cyclodextrin derivative have been practiced in an empirical way so far. In this study, we tried to dev elop a procedure based on MM/3D-RISM method to screen drug can didate cyclodextrin derivative and assessed the capability of our procedure on the test system, for instance, 2-Hydroxypropyl-β-Cyclodextrin and small molecules. To accomplish this objective, we tried to get appropriate ensemble of the complex state and isolated state, and, after that, predict the binding free energy . Our procedure reproduce reasonable correlation between the experimental and calculated binding free energy and we conclude MM/3D-RISM method have an ability to distinguish the tightly bind compounds from the candidate molecules if we get an appropriate ensemble. Now we are applying this procedure to screen a functional cyclodextrin derivative. We are also trying to improve our procedure.
Federal University of Alfenas, Brazil
Time : 16:20-16:40
Ihosvany Camps holds a Bachelor degree in Physics from the Faculty of Physics, University of Havana (Cuba, 1995), Master degree in Physics from the Faculty of Physics, University of Havana (Cuba, 1996) and a PhD in Physics from the Institute of Physics, Federal Fluminense University (Brazil, 2001). He has experience in Condensed Matter Physics and Computational Modelling . Currently, has as m ain research lines the study of electronic properties of nanostructures and the molecular modelling of organic and inorganic systems including the modelling of drugs ( rat ional drug design , fragment-based drug design, and de novo des ign), molecular docking, and studies on polymorphism of pharmaceutical solids.
The molecular docking is the most used technique to theoretically study ligand-receptor interactions . The goal of the tal k is to present the evolution and fundamental aspects behind the molecular docking together with other computational modelling methods capable of giving better information about the ligand-protein interactions than simple docking and ligands with better binding energy . In the first part of the talk, the types of docking (rigid-rigid, rigid-flexible and flexible-flexible) together with its different methods of im plementation (mainly Monte Carlo , Genetic Algorism an d Ant Colony Optimization) are analyzed side-by-side with other techniques like full complex optimization, quantum mechanic polarized ligand docking and fragment molecular orbital. The use and meaning of evaluation fun ctions (score functions) is discussed together with the softwares were they are implemented and they possible relationship with experimental variables. The second part of the talk is dedicated to different methods used to generate new ligands with better interaction (binding) energy to specific targets. Such techniques include fragment-based drug design and de novo design . These techniques generate libraries with thousands of molecules that should be filtered using criteria likes the Tanimoto coefficient of similarity toge ther with ADME (adsorption, distribution, metabolism, excretion) descriptors in order to obtain better drug candidates. We acknowledge financial support of FAPEMIG.
Louisiana State University School of Medicine & Health Sciences Center, USA
Time : 16:40-17:00
Sunyoung Kim has completed her PhD at the University of Michigan and the University of Padova, Italy and Post-doctoral studies at the University of Minnesota. She is a Member of the Louisiana Cancer Research Consortium and Founder of a spin-out company to personalize medicine with structural biomarkers. She leads a collaborative, multilab nanomotor research program. She has published more than 25 papers and serves as an Editorial Board Member of the Journal of Biological Chemistry. In addition, she has performed a variety of administrative roles at the departmental, institutional and state levels, as well as held elected positions for national scientific societies.
ATP hydrolysis requires that a proton f rom the water nucleophile must be abstr acted and transferred in order to create a hydroxide capable of attacking the substrate. In crystallographic capture of ATP hydrolysis, a two-water cluster is found in the active site of two diffe rent kinesin isoforms. These data suggest that a proton is shared between the lytic water , positioned for gamma-phosphate attack, and the second water that serves as a general base. The unusual short distance between the two orthosteric water molecules , observed by crystallography, is confirmed by solvent kinetic isotope experiments. The positive kinetic isotope e ffect (KIE) confirms proton abstraction from water commits kinesin to catalysis and its pH-dependence verifies that switch salt-bridge residues direct chemotransduction . Additionally, a classical descr iption for this proton transfer is refuted by the KIE magnitude, tempe rature-independent Arrhenius pre-exponential fa ctor rati os, and activation energy differences. Taken together, we conclude that the first step in kinesin catalysis has a tunneling component, a quantum mechanical event by which a particle transfers through a reaction barrier. This first detection of tunneling in an ATPase is of consequence for two reasons. First, proton tunneling is likely widespread in biomolecules, rather than solely a characteristic of metalloenzymes. Second, energy barrier penetration by proton tunneling is an alternate explanation to classical transition-state stabilization theory for the fast reactivities of motor proteins.
Tanta University, Egypt
Title: Molecular docking of potent inhibitors of oncoproteins using Swiss dock and visualization by UCSF chimera
Time : 17:00-17:20
Mohammed A. Mansour has completed his PhD at the age of 28 years from Nagoya University graduate school of Medicine , Japan in the field of Biochemistry of cancer and cell signaling . He has b een ass igned the position of lecturer of Biochemistry and Cancer Biology in Tanta University faculty of Science, Egypt since October 2015. He has published up to 10 papers in highly reputed journals including oncotarget, FEBS J, Exp Cell Res and Tumor Biol. He is interested in understanding the molecular pathogenesis of human solid, hard -to-cure cancers. During his PhD project, he focused on the identification of alterations of tumor suppressor genes and oncogenes in colon cancer, followed by elucidation of their detailed biological and biochemical functions. This detailed work res ulted in 3 first author publications, and two co-author papers from collaborative studies. His research thus focuses on taking clinically relevant targets, understanding their molecular function, and determining how to modulate them in the laboratory and in in vivo models.
Histone deacetylases (HDACs) pla y critical roles in apoptosis and contribute to t he proliferation of cancer cells. AR-42 is a novel Class I and II HDAC inhibitor that shows cytotoxicity ag ainst multiple human cancer cell lines. Recently, it was reported that AR-42 inhibits the activity of HDAC5 in hepatocellular carcinoma (HCC). However, the binding mode of action and molecular docking of this inhibitor is not reported. This study aims to evaluate the possible therapeutic efficacy of this inhibitor by testing its dock ing with different targets of HDACs. HDAC5 was found to be upregulated in HCC tissues compared to adjacent normal tissues, and this was correlated with reduced patient survival. Treatment with AR-42 decreased HCC cell growth and increased caspase-dependent apoptosis, and this was rescued by HDAC5 overexpression. We demonstrated that AR-42 can inhibit t he deacetylation ac tivity of HDAC5 by assessing the docking using Swiss dock software. SwissDock is a web service to predict the molecular interactions that may occur between a target protein and a small molecule. Then, we visualized our docking results using UCSF Chimera , a highly extensible, interactive molecular visualizatio n and analysis system. Chimera can read molecular structures and associated data in a large number of formats, display t he structures in a variety of representations, and generate highquality images and animations. Taken together, these results demonstrate for the first time that AR-42 targets HDAC5 and induces its inhibition in human hepatocellular carcinoma . AR-42 therefore shows potential as a new drug candidate for HCC therapy.
Xi’an Jiaotong University, China
Shengli Zhang has completed his PhD at the age of 28 years from Lanzhou University. He is the director of Physics Department at Xi’an Jiaotong University, a top ten university in China. He has published more than 100 papers in reputed journals. His current researches focus on the determination of protein structures and functions by using molecular dynamics simulaitons and electron miscroscopy.
Cholesteryl ester transfer protein (CETP) decreases the level of atheroprotective high-density lipoprotein cholesterol (HDL-C) while elevating the level of atherogenic low-density lipoprotein cholesterol (LDL-C). Several CETP inhibitors have been evaluated in large-scale clinical trials for treating cardiovascular diseases (CVDs) within the last decade. Although the structure of mutated CETP has been revealed through electron microscopy (EM) and the X-ray crystallography, the structure of native CETP in physiological conditions and the detailed mechanism of how CE transfers from HDL to LDL at atomic resolution remains unclear. Here, we employed all-atom molecular dynamics simulations to study the native CETP structure and CE transfer mechanism. We propose the structure of native CETP, which is more difference in some aspects with the crystal structure. The simulation results showed that the tunnel is sufficiently large to mediate the transfer of a CE molecule through CETP with a predicted transfer rate comparable to physiological measurements. Analyses of the interactions and energies between the CE and CETP tunnel during transfer indicated several residues that might regulate CETP function during CE transfer. This study provides insight into the CE transfer mechanism for future development of CETP inhibitors.
- Track 8: Complexity Arenas in Structural Biology | Track 9: Structural Biology in Cancer