Scientific Program

Conference Series Ltd invites all the participants across the globe to attend 9th International Conference on Structural Biology Zurich, Switzerland.

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Day 1 :

Structural Biology 2017 International Conference Keynote Speaker Henry M Sobell photo
Biography:

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.

Abstract:

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.

Keynote Forum

Tilman Schirmer

University of Basel, Switzerland

Keynote: Structural biology of c-di-GMP mediated signaling
Structural Biology 2017 International Conference Keynote Speaker Tilman Schirmer photo
Biography:

Abstract:

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.

Keynote Forum

Shigeyuki Yokoyama

RIKEN Structural Biology Laboratory

Keynote: Structures and functions of seven-transmembrane helix receptors
Structural Biology 2017 International Conference Keynote Speaker Shigeyuki Yokoyama photo
Biography:

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.

Abstract:

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.

  • Special Session: Structural Biology & Single Molecules

Session Introduction

Yuri L. Lyubchenko

University of Nebraska Medical Center, USA

Title: Nanoscale structure and dynamics of centromere nucleosomes

Time :

Speaker
Biography:

Yuri L. Lyubchenko is Professor of Pharmaceutical Sciences at the University of Nebraska Medical Center, Omaha, NE, USA. His research focuses on understanding fundamental mechanisms underlying health and disease, which are key to developing new and more effective diagnostics and medications. This primarily basic research allows him not only identify new drug targets for small molecule drugs, it also leads to development of the nanotools and methods to discover novel approaches for diagnostic, treatment and disease prevention and to more rapidly determine their efficacy at the molecular level.

Abstract:

Statement of the Problem: Chromatin integrity is crucial for normal cell development. The cell division process is accompanied by the segregation of replicated chromosome, and chromatin centromeres, specialized segments of chromosomes provide the accuracy of the chromosomal segregation. If the centromere becomes damaged or removed, chromosomes segregate randomly disrupting the cell division process. The centromeres are specifically recognized by kinetochores suggesting that centromeres contain specific structural characteristics. However, these structural details and the mechanism underlying their highly specific recognition remain uncertain. Methodology & Theoretical Orientation: In this study, we performed direct imaging of CENP-A nucleosome core particles by time-lapse high-speed atomic force microscopy (AFM), enabling us to directly visualize the dynamics of CENP-A nucleosomes. Nucleosomes used for evaluation of DNA wrapping around the histone core were assembled on a DNA substrate containing a centrally positioned 601 motif. Findings: A broadly dynamic behavior of the DNA flanks was first revealed by analysis of AFM images acquired in ambient conditions. Time-lapse imaging further identified the distinctive pathways unique to CENP-A-nucleosome dynamics that are not shared by H3. The spontaneous unwrapping of DNA flanks can be accompanied by the reversible and dynamic formation of loops with sizes equivalent to a single wrap of DNA. Translocation of CENP-A nucleosomes was observed, with the formation of internal DNA loops along the nucleosome. This process was reversible, settling the core back to its starting position. Additionally, the transfer of the histone core from one DNA substrate to another was visualized, as well as distinctive splitting into sub-nucleosomal particles that was also reversible. Conclusion & Significance: Altogether, our data suggest that unlike H3, CENP-A is very dynamic, permitting its nucleosome to distort freely and reversibly, which in turn allows a longer term stability, which may play a critical role in centromere integrity during mitosis and replication.   

Speaker
Biography:

Chromatin is the ubiquitous protein-DNA complex that forms the structural basis of DNA condensation in all eukaryotic organisms. Packaging and depackaging of chromatin, called chromatin remodeling, plays a central role in all cellular processes that involve chromosomes such as transcription, replication, recombination and repair. Detailed knowledge of the principles and mechanisms underlying this control of DNA condensation is thus vital for understanding many diseases, including neurological disorders and cancer. The physical mechanisms governing these processes however, are still largely unknown. I am interested in developing and using modern biophysical techniques to unravel the physics behind DNA condensation and its role in transcription regulation.

Abstract:

The folding of chromatin defines access to our genes and therefore plays a pivotal role in transcription regulation. However, the structure of chromatin fibers is poorly defined and heavily debated. We used single-molecule techniques to probe and manipulate the dynamics of nucleosomes in individual chromatin fibers. These novel methods were initially applied to synthetic, highly homogeneous nucleosomal arrays and yielded unprecedented insight in the structure and dynamics of chromatin.

With single pair Forster Resonance Energy Transfer we showed that the nucleosome is very dynamic, unwrapping half of its DNA four times per second. Using single molecule force spectroscopy, it was possible to measure the kinetics of this unfolding, both in single nucleosomes and in well-defined arrays of nucleosomes that fold into a 30 nm fiber. Analysis of the unfolding pattern reveals a linker length dependence of the higher order folding.

The linker length in vivo however varies, and to obtain insight the positioning of nucleosomes we developed a simple statistical physics model that captures sequence dependent positioning effects for both reconstitutions on synthetic DNA and chromatin in vivo.

We recently developed a method to purify specific chromatin fragments from yeast without crosslinking the fiber while maintaining the complexity that provides functionality to our epi-genome. I will show the first single-molecule force spectroscopy results on intact, native fibers which uniquely probe chromatin structure, composition and variations in it at the single-molecule level.

Speaker
Biography:

Peter Hinterdorfer performs advanced nanoscopic techniques in nano-bio technology, life science, and medical diagnostics, and has been working on antibody/antigen interactions, transmembrane transporters, virus/membrane interactions, cells of the immune system, nuclear envelope membranes, and bacterial surface layers. He has done pioneering work in single molecule force spectroscopy and has invented a combined topography and recognition imaging technique. Recently he did research with high-speed bio-AFM.

Abstract:

In molecular recognition force microscopy (MRFM), ligands are covalently attached to atomic force microscopy tips for the molecular recognition of their cognitive receptors on probe surfaces1. Interaction forces between single receptor-ligand pairs are measured in force-distance cycles. The dynamics of the experiment is varied2, which gives insight into the molecular dynamics of the receptor-ligand recognition process and yields information about the binding pocket, binding energy barriers, and kinetic reaction rates3. Combination of high-resolution atomic force microscope topography imaging with single molecule force spectroscopy provides a unique possibility for the localization of specific molecular recognition events4. The identification and visualization of receptor binding sites on complex heterogeneous bio-surfaces such as cells and membranes are of particular interest in this context4. Considered as the paradigm for molecular recognition are antibodies. They are key molecules for the immune system of vertebrates. The Y-shaped antibody type IgG exhibits C2-symmetry; its Fc stem is connected to two identical Fab arms, binding antigens by acting as molecular callipers. Bivalent binding of the two Fab arms to adjacent antigens can only occur within a distance of roughly 6 to 12 nm. AFM cantilevers adorned with an antibody can measure the distances between 5-methylcytidine bases in individual DNA strands with a resolution of 4Å, thereby revealing the DNA methylation pattern6, which has an important role in the epigenetic control of gene expression. Moreover, due to their nano-mechanical properties antibodies exhibit “bipedal” walking on antigenic surfaces7. The walking speed depends on the lateral spacing and symmetry of the antigens. Importantly, the collision between randomly walking antibodies was seen to reduce their motional freedom. It leads to formation of transient assemblies, which are known to be nucleation sites for docking of the complement system and/or phagocytes as an important initial step in the immune cascade.      

Speaker
Biography:

Gijs Wuite obtained his PhD in biophysics in 2000. Since 2001 he leads his own group at the VU University Amsterdam and in 2009 was appointed to full professor. In his research he has successfully applied quantitative physical tools to investigate fundamental problems in biology, and to search for the unification of apparently unrelated biological phenomena. Moreover, he has been at the front of recent new and fast developments of biophysical techniques that have enabled visualization, manipulation and control of complex biological reactions. Based on this research work he founded in 2014 a company (LUMICKS) that sell the technology he and his group has developed. His work has appeared in journal such as Nature, Science, PNAS and Physical Review Letters. His research has been awarded with the prestigious personal VIDI, VICI and ERC grants. In 2009 Wuite was appointed member of the Young Academy, an independent platform of young top scientists within the Royal Netherlands Academy of Arts and Sciences.

Abstract:

Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells1 . Such breaks are formed, for example, during gene-segment rearrangements in the adaptive immune system or by cancer therapeutic agents. Although the core components of the NHEJ machinery are known, it has remained difficult to assess the specific roles of these components and the dynamics of bringing and holding the fragments of broken DNA together. The structurally similar XRCC4 and XLF proteins are proposed to assemble as highly dynamic filaments at (or near) DSBs2 . Here we show, using dualand quadruple-trap optical tweezers combined with fluorescence microscopy, how human XRCC4, XLF and XRCC4–XLF complexes interact with DNA in real time. We find that XLF stimulates the binding of XRCC4 to DNA, forming heteromeric complexes that diffuse swiftly along the DNA. Moreover, we find that XRCC4–XLF complexes robustly bridge two independent DNA molecules and that these bridges are able to slide along the DNA. These observations suggest that XRCC4–XLF complexes form mobile sleeve-like structures around DNA that can reconnect the broken ends very rapidly and hold them together. Understanding the dynamics and regulation of this mechanism will lead to clarification of how NHEJ proteins are involved in generating chromosomal translocations (Brouwer et al., Nature, doi:10.1038/nature18643 , 2016)  

  • Track 1: Recent Advances in Structural Biology
Speaker
Biography:

John (Ioannis) Vakonakis did a PhD in Biochemistry at Texas A&M University, where he pioneered the structural analysis of bacterial circadian clock proteins. His postdoctoral work at the University of Oxford focused on the structural mechanisms underpinning cell adhesion and assembly of the extracellular matrix in animals. John did breakthrough work on the molecular architecture of the centriole organelle during a second postdoc at the Swiss Light Source, prior to starting his own lab in Oxford Biochemistry. He has been a Marie Currie Fellow, Junior Research Fellow at Trinity College, Oxford, and a Wellcome Trust Research Fellow. John is now Associate Professor in Structural Biology and Biophysics at the University of Oxford, and Fellow in Biochemistry at Lincoln College. Over the last six years his research aims to understand how large molecular machines form in cells, such as the cytoadherence assemblies created upon P. falciparum-infection of human erythrocytes.

Abstract:

Human red blood cells infected by the malaria parasite Plasmodium falciparum (iRBC) form dome-shaped ~120 nm-diameter protrusions on their surface, known as ‘knobs’. Knobs provide essential presentation platforms for the parasite cytoadherence receptor family PfEMP1, which binds ligands on endothelial cells of the blood vessel wall thereby immobilizing iRBC in the microvasculature. The resulting obstruction of blood vessels and disruption of normal circulation causes inflammation and tissue damage that can lead to coma and death. iRBC cytoadherence constitutes the primary mechanism driving morbidity and mortality in P. falciparum infections, which account for over 90% of all malaria-related deaths.

 

Despite their importance in malaria pathology the molecular mechanisms underpinning knob formation remain poorly understood. Here, I review recent progress in characterizing knob complexes formed between parasite and parasite – host proteins. Extensive flexibility is common among parasite knob components, which necessitated an integrative approach to resolve these complexes. In particular, I will focus on the development of novel in silico docking tools suitable for evaluating interactions between folded components and highly charged, very long and flexible protein segments. Our work offers the first glimpse of a molecular model for knobs.

Speaker
Biography:

David Sargent obtained his PhD in biophysics from the University of Western Ontario, Canada, followed by postdoctoral studies at the ETH Zurich and the University of Sydney (Australia). He has had extensive experience in macromolecular crystallography at the ETH Zurich, and recently has also been associated with the Multiscale Robotics Laboratory (ETH Zurich) of Bradley J. Nelson. David is one of the founders of MagnebotiX, a spinoff of the ETH, which provides tools for magnetic propulsion and guidance at the microscopic scale. The work reported here uses this technology to streamline and accelerate the process of macromolecular crystal structure determination. 

Abstract:

Statement of the Problem: Most aspects of macromolecular structure determination, from synthesis and purification of materials, through crystallization, data collection and model building, are highly automated, but the recognition, harvesting and cryocooling of crystals remains a predominantly manual task. Several concepts, including in situ crystallography, are being developed to overcome these difficulties, but frequently impose other restrictions, such as on data collection strategies. We are developing hardware and software to support crystal harvesting using standard crystallization procedures, thus avoiding such limitations. Methodology & Theoretical Orientation: We use a magnetically driven, mobile, rolling microrobot, the “RodBot”, to locally move the liquid surrounding a crystal, and the crystal then passively follows the flow. Crystal position is monitored using low level uv-light. Transport is controlled using flexible algorithms that allow for error-recovery following stochastic disturbances. Findings: We demonstrate the effectiveness of the technique using crystals of different geometries and densities in a variety of buffers and cryoprotectants. Even at this developmental stage average harvesting time is reduced compared to manual operations. Conclusion & Significance: This non-destructive, non-contact method allows crystals to be extracted reliably from the growth droplet in a completely automated process. Harvesting can take place remotely in climate-controlled chambers, ensuring optimal conditions throughout the process with respect to temperature, humidity and composition of the environment. Damage to valuable crystals due to operator jitter or fatigue is eliminated. Incorporation into existing robotics setups for sample handling will also allow increased reproducibility of flash-cooling. Fully automated structure determination pipelines using well-established techniques are now possible and will yield improved data quality at reduced cost.

Speaker
Biography:

Thomas Braun received his Ph.D. 2002 in biophysics from the Biozentrum, University of Basel, Switzerland. During his Ph.D. thesis he applied high-resolution electron microscopy and digital image processing to study the structure and function of membrane proteins. Subsequently, he worked on nanomechanical sensors to characterize the mechanics of membrane proteins at the Institute of Physics, University Basel and the CRANN, Trinity College Dublin, Ireland. He has been working at the Center for Cellular Imaging an NanoAnalytics (Biozentrum, University of Basel, Switzerland) since 2009 and is developing new methods for electron microscopy, single cell analysis and nanomechanical sensors for biological applications.

Abstract:

Cryo-electron microscopy (cryo-EM) sample preparation techniques ensure that biological specimens can be investigated at physiological conditions in the electron microscope.  However, these preparation methods suffer from extensive blotting steps leading to a massive loss of sample and sometimes to partial denaturation of sensitive protein complexes. We have developed a simple method for the almost lossless conditioning and preparation of nanoliter-volumes of biological samples for EM. The method does not involve any blotting steps. A microcapillary is used to aspirate 3 to 20 nanoliters of sample, depending on the experiment (Figure 1A). The sample is applied (left) and spread (center) on the EM-grid. Real-time monitoring allows the thickness of the water film to be assessed and decreased to the optimum value prior to vitrification (right). We prepared cryo-EM grids of various samples, e.g., bacteriophages and soluble proteins as shown in Figure 1B&C, to demonstrate the usefulness and general applicability of the method. We also showed that high-resolution 3D structures can be calculated from single-particle preparations of a soluble protein. In addition to cryo-EM grid preparation, the versatile method allows nanoliter-sized sample volumes to be conditioned for EM, e.g., negatively stained with heavy metal salts or embedded in trehalose.

In addition, we combine the new sample preparation method with a single cell lysis device for adherent eukaryotic cells and image the aspirated cell contents by TEM. To demonstrate the usefulness of this new “visual proteomics” approach we visualized the changes occurring in single cell proteomes upon heat shocking the cells. Furthermore, we have developed a protein-fishing method based on a magnetic trap and photo-cleavable composite material, to ‘fish’ untagged proteins from cell lysate by antibodies. This allows target proteins to be isolated from approx. 40’000 cells in 90 min and analysed by EM. 

Irena Roterman

Jagiellonian University – Medical College, Krakow, Poland

Title: Influence of water environment on protein structure

Time :

Speaker
Biography:

Irena Roterman-Konieczna heads the bioinformatics group at the Jagiellonian University Medical College and the Faculty of Physics, Astronomy and Applied Computer Science, Poland. Her background is the theoretical chemistry. She worked for Harold Scheraga group as the postdoc (Cornell University – Ithaca NY in 1987-1989). Her research focuses on bioinformatics with a specific orientation on protein structure prediction. Particularly the application of fuzzy oil drop model (Irena Roterman and Leszek Konieczny are the Authors) appears to be successful for 3D protein structure analysis including the active site recognition, protein-protein complexation and recently for recognition of amyloidogenesis mechanism. 

Abstract:

Model to represent the influence of water environment on the protein structure is expressed by fuzzy oil drop model. The main assumption is that the idealized distribution of hydrophobicity in protein molecule can be expressed by 3D Gauss function (Fig.1. – blue line). According to this function the protein molecule encapsulated in ellipsoid represents the highest hydrophobicity in the center of molecule with gradual decrease together with the increase of distance versus the center reaching the zero-level on the surface of molecule in the distance of 3sigma in any direction. This organization of hydrophobicity makes the protein molecule perfectly well soluble however deprived of any form of activity. Activity requires the local elasticity (fluctuations) and generation of local force field for ligand (substrate) to be bound to protein molecule. This is why the local discrepancies versus the idealized hydrophobicity distribution are observed mainly in the area of biological activity. The discrepancies - local hydrophobicity excess or local hydrophobicity deficiency is interpreted as coded form for protein-protein complexation (mediated by exposed hydrophobicity) in the first case and ligand binding area in the second.

The local discrepancy versus the idealized distribution can be measured quantitatively applying the Kullback-Leibler entropy formula expressing the distance between two distributions: target one (idealized 3D Gauss function distribution) and the observed one as it appears in certain protein molecule (inter-residual hydrophobic interaction). This measurement allows comparison of structures of high topological similarity with however high dissimilarity in a sense of hydrophobicity distribution. This was shown in the cease of immunoglobulin and flavodoxin families. The fuzzy oil drop model was successfully applied for the construction of the model of amyloidogenecity. The encapsulation of the protein molecule in the shell of 3D Gauss function form ensures the generation of individual soluble units. The specific disorder versus the ordered one generates hydrophobicity distribution supporting the propagation of omplexation in one-dimensional form of fibrils.  

Speaker
Biography:

Dino Moras, PI, graduated in chemistry at the University of Strasbourg. While post-doc with M.G. Rossmann he contributed to the concept of nucleotide binding domain known as the 'Rossmann fold'. His main scientific contributions are in structural biology, related to the expression of the genetic information: (i) translation of the genetic code by aminoacyl-tRNA synthetases: discovery of the partition of aaRS in two classes and first crystal structure of a class II tRNA-aaRS complex (ii) transcription regulation by Nuclear Receptors: the first crystal structures of the ligand binding domains of two NRs (RXR and RAR) in their apo and liganded form respectively. Presently his main focus is on the molecular mechanisms of regulation using integrative structural biology approaches.

Abstract:

Nuclear Hormone Receptors interact with corepressors, coactivators and other protein cofactors to regulate signal transduction of the basal transcriptional machinery. Most NRs are known to function as dimers and with the exception of the group of oxosteroid receptors (AR, GR, MR, PR) all structural data point to a conserved interface for the ligand binding domains (LBDs) dimers.

Allosteric mechanisms control the sequential and ordered binding of nuclear receptors to the various protein effectors and target DNA. The binding of ligands induce structural transitions in the LBDs leading to the release of the corepressors and their replacement by cofactors. The LBD swallows the ligand and shields it from the solvent by closing the pocket with the C-terminal peptide. The agonist/antagonist character of the ligand is then essentially controlled by the position of helix H12 and the stability of the complex. Ligands are modulators of the activation process, their potency being defined by the fraction of time spent in the active conformation. Crystal structures of DNA binding domains (DBDs) bound to different response elements also support the proposal of DNA being an allosteric effector. The architectures of full length receptors bound to DNA fragments and cofactors have been determined by crystallography and in solution using integrative approaches. The later combine structural small angle diffraction methods by X-Rays (SAXS) and neutrons (SANS), optical techniques like FRET with labelled molecules and single particle electron microscopy (cryo-EM). Some common features emerge that rationalize the key role of DNA. The recent advances in cryo-EM allow solution structures determination at near atomic resolution. Conformational equilibrium of NRs in complex with various cofactors are also accessible.

Speaker
Biography:

Robert Craigie is a Senior Investigator in the National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health, Bethesda, MD, USA. His research has focused on the mechanism of retroviral DNA and the structure and function of proteins and nucleoprotein complexes that mediate it.

Abstract:

Statement of the problem. Integration of retroviral DNA into host DNA is an essential step in the replication of HIV-1 and other retroviruses. Integration is mediated by a nucleoprotein complex (intasome) comprising the virally encoded integrase enzyme and a pair of viral DNA ends. The first intasome on the integration pathway is the stable synaptic complex (SSC) in which a pair of viral DNA ends is bridged by integrase. Within the SSC, integrase then cleaves two nucleotides from the 3’ ends of the viral DNA to form the cleaved stable synaptic complex (cSSC). The cSSC captures a target DNA and a pair of transesterification reactions covalently joins viral to target DNA. Currently approved inhibitors of HIV-1 DNA integration target intasomes (specifically the cSSC) rather than free integrase protein, High-resolution structures of intasomes are required to understand their detailed mechanism of action and how HIV-1 can escape by acquiring resistance. Methodology and strategy. Although the structures of the individual domains of HIV-1 integrase were determined more than two decades ago, attempts to obtain high-resolution structures of HIV-1 intasomes were unsuccessful. The main obstacles were the the propensity of both integrase and intasomes to aggregate and the low efficiency of assembly in vitro. We have overcome these problems by developing a hyperactive integrase mutant that assembles intasomes that are amenable to biophysical and structural studies. CryoEM studies of STCs reveal both tetrameric and higher order species that both share a common core architecture with intasomes of related retroviruses. SSCs also assemble both tetrameric and higher order intasomes and both are active for concerted DNA integration in vitro. Conclusions and significance. The results highlight how a common core intasome architecture can be assembled in different ways. Structures of cSSC intasomes in complex with inhibitors will elucidate their detailed mechanism of action and mechanisms by which HIV-1 can evolve drug resistance.

Speaker
Biography:

Nebojsa Janjic has been Chief Science Officer at SomaLogic, Inc. since January 2009. Prior to joining SomaLogic, Dr. Janjic was a founder and CSO at Replidyne, Inc., a biotechnology company focusing on the development of new small-molecule antibacterial agents. Prior to Replidyne, Dr. Janjic was senior director of drug discovery at NeXstar Pharmaceuticals, where his contributions include the discovery and early development of Macugen, the first aptamer to receive FDA approval and the first VEGF inhibitor developed for the treatment for macular degeneration. As CSO at SomaLogic, Dr. Janjic is involved in developing a new generation of modified aptamers and identifying opportunities for their use in science and medicine. Dr. Janjic received his bachelor's degree in molecular biology and Ph.D. in physical organic chemistry from the University of Washington in Seattle and completed his postdoctoral training at the Scripps Research Institute in La Jolla as a Cancer Research Institute Fellow.

Abstract:

Statement of the Problem: The ability to fold into distinct three-dimensional structures is the basis of high affinity and specificity characteristic of aptamer binding to their targets. We have recently introduced base modifications that increase chemical diversity of functional groups front-loaded in randomized nucleic acid libraries from which aptamers are selected. Such modifications have allowed us to identify high-affinity aptamers to a large number of protein targets previously considered “difficult” with conventional nucleic acid libraries. At the same time, our ability to predict the structures of modified aptamers with conventional nucleic acid folding rules was severely compromised, suggesting new rules for folding. Methodology & Theoretical Orientation: We examined published structures of sixteen aptamers co-crystallized with their protein targets, including three aptamers with base modifications we reported recently. Findings: In contrast to small molecules, which are entirely encaged by aptamers, proteins present large surfaces with distinct features that are recognized by complementary surfaces on aptamers. The size of these interaction surfaces is comparable to those observed with antibodies, although for aptamers, the size range is wider on both small and large extremes. The highly flexible phosphodiester backbone allows assembly of known as well as novel nucleic acid motifs into precise three-dimensional structures that orient often discontiguous aptamer regions toward their protein targets in a manner that creates surfaces with exquisite shape complementarity. Base modifications with hydrophobic side chains allow occupancy of distinctly hydrophobic pockets on proteins and create novel structural elements that illustrate the profound role modified nucleotides play in both folding and binding. Conclusion & Significance: These observations provide compelling structural rationale for the observed high affinity and specificity with which aptamers recognize their protein targets, and show us that the lexicon of structural features accessible to nucleic acid ligands can be vastly expanded with chemical modifications of nucleic acid libraries.

Toshiya Senda

High Energy Accelerator Research Organization (KEK), Japan

Title: The native (Sulfur) SAD method in Photon Factory
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 (KEKI) 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:

Crystallography has been a major method to determine 3D structures of biological macromolecules at atomic resolution. While a new method with cryo-EM is becoming another major technique for the 3D structure analysis, crystallography still has some advantages. Recently, many crystal structures of biological macromolecules are determined by MAD/SAD method with seleno-methionine proteins (SeMet-proteins). Since selenium has an X-ray absorption edge near 1Å, it is convenient to utilize in the MAD/SAD method. While this technique is useful, crystallographers need to prepare SeMet-proteins. If we can develop a method to solve the phase problem without using SeMet-proteins, it would be highly useful for crystallographers. So, we have tried to develop the native SAD (or sulfur SAD) method, in which anomalous signals from sulfur atoms in the native protein are utilized. However, there are some problems in the native SAD method. First, sulfur gives only weak anomalous signals with X-ray typically utilized in protein crystallography (X-ray wavelength of around 1Å). To increase the anomalous signals, we need to use a longer wavelength X-ray than usual. However, since a longer wavelength X-ray shows significant absorption by air, solvent, protein etc., data quality is degraded by the absorption. The native SAD method, therefore, requires a specific system for high quality data collection. To achieve routine utilization of the native SAD method, we have developed a beamline (BL-1A) dedicated for the native SAD method. In BL-1A, we can utilize a long wavelength X-ray (1.9 – 3.5 Å). Furthermore, the goniometer and X-ray detector are installed inside a He chamber to prevent the absorption problem. Our system enables us to solve crystal structures of proteins by the native SAD method. In the presentation, we will present several examples of crystal structure determination with native SAD. Also, we will mention our unique method for crystal freezing to collect high quality diffraction data required in native SAD experiments.

Biography:

Julien Boudet received his PhD degree in structural biology and biophysics from the University of Grenoble (Joseph Fourier University) in France under the supervision of Prof. Jean-Pierre Simorre. During his thesis, he learned nuclear magnetic resonance (NMR) spectroscopy and used this powerful method to investigate proteins and oligonucleotides structures, molecular mechanisms underlying antibiotic resistance and viral proteins interactions. After graduating, Julien joined the group of Prof. Frédéric Allain in ETH Zurich as a researcher. He focused his investigations on the DNA replication machinery and, in particular on the primase-mediated catalysis. He set up innovative computational methods to investigate challenging biological systems and demonstrated the role of cofactors in improving the pRN1 primase specific template recognition. His currently developing computational and analysis tools for structural biology.

Abstract:

Primases are single-stranded DNA dependent polymerases that synthesize RNA/DNA primers during replication. A primase, a DNA polymerase and an helicase compose the replication machinery of the archaeal plasmid pRN11. The structure of the archaeal functional primase domain has been solved by X-ray crystallography 2,3 and it revealed an heteromeric structure with a catalytic prim/pol domain tethered to a novel helix bundle domain.

We investigated the NMR structure of the functional pRN1 primase domain in complex with a single-stranded DNA template containing the GTG motif 4. We showed that the catalytic prim/pol domain of this 38 kDa enzyme is not required for template binding. Intermolecular contacts detected exclusively between the helix bundle domain and the DNA led us to isolate specifically this structurally independent unit. Our results are compatible with a conformational switch between a template-bound open state and a closed active complex 3,5,6.

We solved the solution structures of the helix bundle domain in complex with the single-stranded DNA template alone and upon cofactors addition. Affinity measurements validated our structural data demonstrating the importance of residues located in helices 10 and 12 for the interaction with the GTG motif and confirmed the specificity improvement observed upon cofactors binding.

In association with functional assays, these novel transient structures bring new perspectives and will help us to characterize the molecular steps required for priming.

Speaker
Biography:

Luis Paulo Barbour Scott is Associate Professor in Federal University of  UAFBC.  He has his expertise in conformational changes and functional movements of macromolecules, specially proteins. Over the last four years,  Dr. Luis Paulo Scott’s research group has been financed to investigate molecules related to neurodegeneration and aggregate formation by means of normal mode analysis and molecular dynamics  combined. The laboratory coordinated by Dr. Luis Paulo Scott has become more and more specialized in the study of macromolecules structural dynamics (functional movements in collaboration with Dr. David Perahia from France. 

Abstract:

Human immunodeficiency virus type-1 protease (HIV-1 PR) is an aspartic protease whose proteolytic activity is essential for cleaving precursor viral polyproteins into individual proteins implied in viral replication. Once HIV enters within a host cell, its RNA is transcribed into DNA through reverse transcriptase, integrated and amplified along with the replication of the host cell's DNA. Gag and gag-pro-pol genes are transcribed into messenger RNA, translated into gag and gag-pro-pol precursors proteins in the cytoplasm, and then assembled at the cell surface for budding and formation of the immature viral particles. In this work, we propose a computational protocol to generate and select HIV protease conformations relevant to its function using Normal Mode Analysis (NMA). We have considered structures of the apoenzyme, the protein with its substrate and product and the protein with a drug.  This set of structures should reveal large amplitude motions that are critical to the protease activity cycle as: i) substrate acquisition; ii) substrate cleavage and; iii) product release. The apoenzyme presents an increased flap conformational diversity compared to the various complexes, predominantly populated with open flap conformations, that can possibly be related to the substrates acquisition.  The enzyme-substrate complexes show more structural diversity than enzyme-product complexes, suggesting a role of these conformational changes in catalytic activity. We presents a promising protocol to identify the conformational diversity induced by different types of ligands and that can help the drug design process

  • Track 2: 3D Structure Determination | Track 3: Computational Approaches in Structural Biology | Track 4: Hybrid Approaches
Location:
Speaker
Biography:

Pedro J. de Pablo has his expertise in the application of Atomic Force Microscopy to the investigation of individual viruses. He studied Condensed Matter Physics at the Universidad Autonoma de Madrid and worked with a variety of molecular wires, including carbon nanotubes and DNA during his PhD. Then he moved for a postoc in the Vrije Universiteit to apply AFM to the study of molecular motors (kinesin) and viruses. He stablished his own group back in Madrid, where during the last 10 years has been applying AFM to unveil the structure-function interplay of viruses by measuring thir physical properties, such as mechanics and electrostatics.

Abstract:

The basic architecture of a virus consists of the capsid, a shell made up of repeating protein subunits, which packs, shuttles and delivers their genome at the right place and moment. Viral particles are endorsed with specific physicochemical properties, which confer to their structures certain meta-stability whose modulation permits fulfilling each task of the viral cycle. These natural designed capabilities have impelled using viral capsids as protein containers of artificial cargoes (drugs, polymers, enzymes, minerals) with applications in biomedical and materials sciences. Both natural and artificial protein cages (1) have to protect their cargo against a variety of physicochemical aggressive environments, including molecular impacts of highly crowded media, thermal and chemical stresses, and osmotic shocks. Viral cages stability under these ambiences depend not only on the ultimate structure of the external capsid, which rely on the interactions between protein subunits, but also on the nature of the cargo. During the last decade our lab has focused on the study of protein cages with Atomic Force Microscopy (AFM) (figure 1). We are interested in stablishing links of their mechanical properties with their structure and function. In particular, mechanics provide information about the cargo storage strategies of both natural and virus-derived protein cages (2,3). Mechanical fatigue has revealed as a nanosurgery tool to unveil the strength of the capisd subunit bonds (4). We also interrogated the electrostatics of individual protein shells (5). Our AFM-fluorescence combination provided information about DNA diffusing out cracked-open protein cages in real time (6).

Miki Senda

High Energy Accelerator Research Organization (KEK), Japan

Title: Molecular mechanism of SHP2 activation by CagA from Helicobacter pylori
Speaker
Biography:

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 has 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.

Abstract:

Helicobacter pylori, which is known as a major risk factor of stomach cancer, delivers an effector protein CagA into gastric epithelial cells. CagA then promiscuously interact with host proteins, SHP2 and PAR1b, to deregulate these proteins, potentiating oncogenic signaling. CagA comprises an N-terminal structured region and a C-terminal intrinsically disordered region which interacts with the host proteins. We already determined the crystal structure of the N-terminal region of CagA (1–3). The crystal structure revealed that the basic amino-acid cluster in the N-terminal region is utilized to localized CagA at the inner face of the plasma membrane. After localization, short segments including Glu-Pro-Ile-Tyr-Ala (EPIYA) motif in the C-terminal disordered region are phosphorylated by Src and interact with SHP2 to deregulate its phosphatase activity. In this study, we have analyzed the structure-function relationship of the EPIYA-segments of CagA. Based on the sequence flanking each of the EPIYA motifs, four types of EPIYA segments, A, B, C, and D, have been identified (4). It is already known that combinations of the EPIYA segments are geographically different (Western and East Asian CagA) and affect CagA’s oncogenic activity. While Western CagA with EPIYA-A, B, and C segments has weak oncogenic activity, Western CagA with EPIYA-A, B and multiple EPIYA-C segments shows increased oncogenic activity (5). East Asian CagA, which has much higher oncogenic activity than Western ones, typically possesses EPIYA-A, B, and D in the C-terminal region. Our biochemical data revealed that oncogenic activity of CagA is correlated with binding affinity for SH2 domain of SHP2 (SH2_SHP2). We have analyzed the interaction between SH2_SHP2 and the EPIYA-C/D segment using biochemical, crystallographic, and physicochemical methods and revealed two types of activation mechanisms of SHP2. In our presentation, we will report that East Asian and Western CagA utilize two distinct activation mechanisms of SHP2.

Speaker
Biography:

Kyoko Shinzawa-Itoh, associate Professor of University of Hyogo, grew up in Hiroshima Japan. She received her MS degree from Hiroshima University of Graduate School of Integrated Arts and Sciences and her Ph D. in Pharmaceutical Sciences from Hiroshima University of Graduate School of Biomedical & Health Sciences. She worked as assistant professor at the Department of Life Science, Himeji Institute of Technology 1988-2004 and at the Hyogo University of Graduate School of Life Science 2004-2013. She is associate Professor of Picobiology Institute, Graduate School of Life Science University of Hyogo from 2013. She has studied about mitochondrial respiratory complexes.

Abstract:

Mitochondrial cytochrome c oxidase (CcO) transfers electrons from cytochrome c (Cyt.c) to O2 to generate H2O, a process coupled to proton pumping. To elucidate the mechanism of electron transfer, a crystal structure of the complex of CcO and Cyt.c would be invaluable for mechanistic studies. Two-dimensional (2D) crystals of the mammalian Cyt.cCcO complex were prepared at higher pH (7.4–9.0) with both proteins in the oxidized state (Osuda et al, 2016), but these 2D crystals could not provide us with a structure of sufficient resolution. We optimized 3D crystallization conditions for ferri-Cyt.c and oxidized CcO at high pH and solved the X-ray structure of the complex at 2.0 Å resolution. The specific interaction between Cyt.c and CcO is stabilized by only six electrostatic interactions between side chains within a small contact surface. From a theoretical calculation based on the complex structure, we identified an electron transfer pathway from the heme c of Cyt.c to CuA of CcO via Lys-13 of Cyt.c. Between the two proteins are three water layers, one of which lies between the other two layers without significant direct interaction with either protein. The inter-molecular span between Cyt.c and CcO is longer than those of other complexes by more than 3.0 Å, and the contact surface area of Cyt.c and CcO is smaller than one-third the size of those of other complexes. Cyt.c undergoes large structural fluctuations, using the interacting regions with CcO as a fulcrum. These features of the protein–protein interaction at the docking interface represent the first known example of a new class of inter-protein interaction, which we term “soft and specific”.  This interaction is predicted to contribute to the rapid association/dissociation of the Cyt.cCcO complex, which facilitates the sequential supply of four electrons for the O2 reduction reaction.

Speaker
Biography:

Hyun-Soo Cho’s research aims to understand the structural and functional role of various proteins involved in cancer and immune diseases. He is specialized in X-ray Crystallography to solve protein structures with other biophysical and biochemical techniques including Cryo_EM recently. His ongoing research projects include various enzymes and receptors especially G-Protein Coupled Receptor (GPCR) related with cancer and immune system. 

Abstract:

Recently, light-driven sodium pump rhodopsin (NaR/KR2/NDQ rhodopsin) and chloride pump rhodopsin (ClR/NTQ rhodopsin) from marine flavobacteria were identified by metagenomics study. One of them, light-driven sodium pump rhodopsin (NaR) structure was determined. The other one we have solved the first crystal structure of a unique class light-driven chloride pump (ClR) from Nonlabens marinus S1-08, at resolutions of 1.57 Å. Like structured Halorhodopsin (HR), ClR can transfer chloride ion from extracellular to cytosol. Although both ClR and HR are same light-driven chloride pump rhodopsin, we found some evidences that ClR and HR are different in structure and mechanism. The structures reveal two chloride-binding sites, one around the protonated Schiff base and the other on a cytoplasmic loop. We identify a “3 omega motif” formed by three non-consecutive aromatic amino acids that is correlated with the B-C loop orientation. Detailed CIR structural analyses with functional studies in E. coli reveal the chloride ion transduction pathway. Our results help understand the molecular mechanism and physiological role of ClR and provide a structural basis for optogenetic applications.

Biography:

Jianyong Li is a biochemistry professor at Virginia Tech. He has extensive experience in protein-related studies, including protein expression and purification, protein functional determination and protein structure and function relationships. Particularly worth to mention is the functional establishment of some unique yellow genes in insects and structural and function relationship of enzymes involved in kynurenate synthesis in mammals.

Abstract:

Bacterial effectors are proteins secreted by pathogenic bacteria into host cells through a type 3 or 4 secretion system. These bacterial effectors may help the pathogens to invade host cells and/or suppress its immune system; thereby promoting their infection, survival and reproduction. Effector proteins are primarily responsible for the pathogenicity of a given bacterial pathogen; therefore, learning the specific mechanism but which their effectors enter into host cells may provide insights for disease prevention. Bacterial Type 3 secretion system (T3SS) has been extensively studied. It is a needle-like structure made by a number of structural proteins, which is responsible for transfer of protein effector to host cells. The needle tip is ~ 3 nm, which is smaller than the required dimension for most bacterial effectors.  Despite some better understanding of the T3SS structure, how their bacterial effectors gain entry to host cells remains speculative. Using a T3SS-dependant effector as a model from Xanthomonas oryzae (a bacterium causing serious disease to some essential plants), we determined the structures of an effector protein in complex with a chaperone-like protein. In the genome of Xanthomonas oryzae, the coding sequence of effector protein is adjacent to that coding the chaperone-like protein. Our structural analysis indicates that the effector-chaperon complex crystallized as tetramers (1.64 A resolution). The monomer of the protein effector contains a T4 polynucleotide kinase domain, while the monomer of the chaperon includes a novel kinase binding domain. Our data suggest that the chaperone protein interacts with the protein effector in a manner that helps to stabilize the protein effector and prevents the virulence effect of protein effort from harming the bacteria before being transferred to host cells. Currently, efforts are being made to understand the precise roles the chaperon protein plays during the transfer of protein effector to host cells. 

Speaker
Biography:

The Kolaczkowski Lab uses a combination of computational biology, statistics, structural modeling and biochemistry to examine how molecular systems evolve. We are particularly interested in the evolution of innate immunity in animals and plants. Dr. Kolaczkowski’s research program focuses on developing cutting-edge tools for the characterization of molecular-evolutionary processes and applying these approaches to learn how long-term protein evolution works. Dr. Kolaczkowski’s recent work has advanced the fields of ancestral sequence resurrection (ASR) and protein function prediction while shedding new light on the evolution of innate antiviral immunity and RNA interference.

Abstract:

Determining the evolutionary forces driving transitions in protein function, the structural mechanisms through which functions change and the effects on cellular signaling is central to molecular evolution. These questions are commonly examined using computational and statistical approaches. More recently, ancestral sequence resurrection (ASR) has combined inference of ancient protein sequences with experimental functional characterization. However, the reliance of these studies on low-throughput experiments limits them to examining a relatively small number of hypotheses. Currently, we have little direct, unbiased information about how protein function may change across large phylogenies. Here we develop an approach integrating large-scale ancestral sequence reconstruction with structural modeling, molecular dynamics, function prediction and experimental validation to characterize molecular-functional shifts and their structural bases across protein families with 1000s of sequences. We apply this approach to study two families of RNA-binding proteins spanning animal and plant lineages. We identify discrete shifts in protein-ligand affinities and long-term changes in function occurring across multiple nodes. Our results suggest that changes in protein function may not always be associated with gene duplication or major speciation events. The identification of functional 'flip flopping' - repeated transitions among a small number of functional states through different structural mechanisms - also supports the view that protein function may be highly evolutionarily labile. We characterize co-evolution between RNA binding proteins and their signaling partners, suggesting that co-evolutionary processes may be common, even when large shifts in molecular function are rare. We suggest that an 'unbiased' view of protein functional evolution may reveal new information about how protein families evolve when we aren't looking.

CongBao Kang

Agency for Science, Technology and Research (A*STAR), Singapore

Title: Structural and dynamic studies of DENV and ZIKV proteases and its insight into inhibitor design
Speaker
Biography:

CongBao Kang received his Ph.D. from School of Biological Sciences at Nanyang Technological University (NTU). He was a research fellow at Centre for Structural Biology, Vanderbilt University, where he was working on structural determination of disease-related membrane proteins. He is currently the group leader of high End NMR group at ETC. His group is working on protein structure, dynamics and its interaction with potential drug candidates using solution NMR spectroscopy. The goal of his group is to provide structural information of a target protein to the medicine chemists to understand structure-activity relationship of potent compounds. His group is involving in hits identification, hits to lead, and lead optimization steps of the drug discovery process. His is currently working on target-based drug discoveries. The targets include methyltransferases, kinases, ion channels, membrane-bound receptors, protein-protein interactions, and viral proteins.

Abstract:

Dengue virus (DENV and Zika virus (ZIKV) belong to Flaviviridae genus which contains important human pathogens. DENV affects people living in tropical and subtropical regions. DENV infection can cause serious diseases such as dengue fever. ZIKV has drawn worldwide attention because of the outbreak in 2015. Viral genome of a flavivirus encodes a polyprotein that can be processed into structural and non-structural (NS) proteins by both host and viral proteases. Viral protease is a two-component serine protease formed by a cofactor region (~40 aa) from NS2B and a protease region (~170 aa) from NS3. The NS2B-NS3 protease of DENV or ZIKV is a validated target because of their function in maturation of viral proteins. Structural studies have been conducted for both DENV and ZIKV proteases. For DENV, previous studies have demonstrated that the free protease adopts an open conformation in which the C-terminal part of the NS2B cofactor region stays away from the active site. In the presence of an inhibitor, DENV protease forms a closed conformation in which the C-terminal region of NS2B forms part of the active site and interacts with the inhibitor. Our NMR study reveals that an unlinked DENV protease adopts the closed conformation in solution. Based on the knowledge on DENV protease, several constructs were made for ZIKV protease. Structural studies demonstrated that ZIKV protease adopts the closed conformation in the absence and presence of an inhibitor or substrate. The linker or enzymatic cleavage site present between NS2B and NS3 may affect inhibitor to interact with the active site. Our accumulated studies have shown that the unlinked protease construct can be used for studying protease-inhibitor interactions. We have demonstrated that the unlinked ZIKV protease interacts with different types of inhibitors. Our studies will be helpful for structure-based inhibitor design against both ZIKV and DENV proteases.

Speaker
Biography:

Chromatin is the ubiquitous protein-DNA complex that forms the structural basis of DNA condensation in all eukaryotic organisms. Packaging and depackaging of chromatin, called chromatin remodeling, plays a central role in all cellular processes that involve chromosomes such as transcription, replication, recombination and repair. Detailed knowledge of the principles and mechanisms underlying this control of DNA condensation is thus vital for understanding many diseases, including neurological disorders and cancer. The physical mechanisms governing these processes however, are still largely unknown. I am interested in developing and using modern biophysical techniques to unravel the physics behind DNA condensation and its role in transcription regulation.

Abstract:

The folding of chromatin defines access to our genes and therefore plays a pivotal role in transcription regulation. However, the structure of chromatin fibers is poorly defined and heavily debated. We used single-molecule techniques to probe and manipulate the dynamics of nucleosomes in individual chromatin fibers. These novel methods were initially applied to synthetic, highly homogeneous nucleosomal arrays and yielded unprecedented insight in the structure and dynamics of chromatin.
 
With single pair Forster Resonance Energy Transfer we showed that the nucleosome is very dynamic, unwrapping half of its DNA four times per second. Using single molecule force spectroscopy, it was possible to measure the kinetics of this unfolding, both in single nucleosomes and in well-defined arrays of nucleosomes that fold into a 30 nm fiber. Analysis of the unfolding pattern reveals a linker length dependence of the higher order folding.
The linker length in vivo however varies, and to obtain insight the positioning of nucleosomes we developed a simple statistical physics model that captures sequence dependent positioning effects for both reconstitutions on synthetic DNA and chromatin in vivo.
 
We recently developed a method to purify specific chromatin fragments from yeast without crosslinking the fiber while maintaining the complexity that provides functionality to our epi-genome. I will show the first single-molecule force spectroscopy results on intact, native fibers which uniquely probe chromatin structure, composition and variations in it at the single-molecule level.

Speaker
Biography:

Alfredo De Biasio’s work focuses on the structure and function of DNA sliding clamps and their complexes operating in DNA replication and repair. He is particularly interested in understanding the mechanisms of sliding of the eukaryotic clamp PCNA, and how these mechanisms are modulated by modifications of the PCNA sliding surface, and the implications in DNA damage avoidance. These problems are tackled by an integrative approach that combines X-ray crystallography, NMR and MD simulations.   

Abstract:

Sliding clamps encircle DNA and tether polymerases and other proteins to the genomic template, and are essential factors in DNA replication. Because of the transient interaction that the clamps establish with DNA, the clamp-DNA interface eluded a thorough structural characterization, so that the molecular mechanism for clamp sliding on DNA remained obscure. Here, I will show how the combined use of high-resolution techniques (X-ray crystallography and NMR) and molecular dynamics (MD) simulations allowed to visualize the interactions between the Proliferating Cell Nuclear Antigen (PCNA) – the eukaryotic sliding clamp – and DNA, and to decipher the mechanics of sliding. In addition, recent findings show that the DNA sliding surface of PCNA can be modified to regulate the resistance to DNA damage. From a structural viewpoint, I will reflect on these findings which open a new perspective on PCNA function and offer opportunities to develop tools to manipulate the DNA damage response in cancer treatment. 

Speaker
Biography:

Stanislav Engel, Ph.D., now is an Assistant Professor in the Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, The National Institute for Biotechnology, Ben-Gurion University in the Negev, Beer-Sheva, Israel. He got his B Sc in biochemistry, M Sc and Ph.D. in biochemistry and biotechnology engineering at the Ben-Gurion University in the Negev. Currently, Dr. Stanislav Engel’ researches focus on the understanding the structural basis of “protein misfolding” diseases, such as ALS, and structure-based drug discovery.

Abstract:

Dissecting G protein-coupled receptors (GPCR) signaling in terms of the pathways being activated will boost our understanding of the molecular fundamentals of hormone action. The structural determinants governing the selectivity of GPCR/G protein coupling, however, remain obscure. The selectivity of GPCR/G protein recognition appears to be determined by both specific inter-residue interactions and features related to the overall 3D conformation of the ICD. It appears, therefore, that to elucidate the fundamentals of the selectivity of GPCR/G protein recognition, a comprehensive analysis of the structure-activity relationships of multiple GPCR complexes with different G protein isoforms is required. However, enormous technical difficulties associated with the isolation of functional receptors in quantities required for direct structural studies effectively impede progress in the field. Methodology: We constructed the functional mimetics of the intracellular domain (ICD) of a model GPCR - thyrotropin receptor (TSHR), based on a unique scaffold, 6-Helix, an artificial protein that was derived from the elements of the trimer-of-hairpins structure of HIV gp41 and represents a bundle of six a-helices. Findings: The 6-Helix scaffold, which endowed the substituted TSHR ICD elements with spatial constraints analogous to those, found in native receptors, enabled the reconstitution of a microdomain comprising the intracellular loops ICL-2 and ICL-3, which is capable of binding and activating Ga-(s). Conclusion & Significance: By using a soluble scaffold, which furnishes peptides derived from the GPCR ICD with spatial constraints similar to those, found in native receptors, the reconstitution of a native-like G protein-recognition epitope can be facilitated. The 6-Helix-based mimetics could be used as a platform to study the molecular basis of GPCR/G protein recognition. Such knowledge could lead to the development of novel therapeutic strategies for GPCR-related disorders by targeting the GPCR/G protein interfaces and help counteract cellular dysfunctions via focused tuning of GPCR signaling.

 

  • Track 5: Molecular Modeling
Location:
Speaker
Biography:

Christina Scharnagl has her expertise in molecular dynamic simulations of membrane proteins. Her work focuses on biophysical principles of the interdependence of transmembrane helix dynamics, helix-helix binding, and helix-lipid interactions. In silico modelling and advanced computational analysis are closely connected to experimental work in research collaborations in order to interpret and guide the experiments and to validate the simulations. The aim of the joint efforts is to understand the impact of these phenomena on multiple biological processes, such as membrane fusion and intramembrane proteolysis. 

Abstract:

Integral membrane proteins facilitate communication between the inside of the cell and its exterior. Their transmembrane domains (TMDs) support a diversity of biological functions and exhibit sequence-dependent conformational dynamics on multiple size and time scales. Membrane proteins are notoriously difficult to study by experimental methods. Molecular dynamics (MD) simulations provide a powerful tool of high spatial and temporal resolution that effectively complements experimental methods. Here we focus on the conformational dynamics of the TMD of the amyloid precursor protein (APP). APP is enzymatically hydrolyzed within its TMD by g-secretase (GSEC), forming toxic Ab peptides regarded as molecular cause of Alzheimer's disease (AD). Besides APP, GSEC cleaves ~100 single-span membrane proteins within their TMDs, however without obvious consensus sequence. Finding the link between the molecular architecture of the substrate TMDs and cleavage is, therefore, of upmost importance. Because unfolding is obvious to expose the scissile bond, it seems plausible that the TMD itself is optimized for local helix unwinding. However, this view was challenged by our experiments and MD simulations. Our results suggest an alternative model where reaching a cleavage-competent state involves multiple conformational transitions of the substrate/enzyme complex where global conformational plasticity of the substrate TMD is a key determinant. In a first step, we compare the conformational flexibility of a large number of substrate and non-substrate TMDs, as well as TMDs carrying missense mutations related to early onset AD. Knowing the key-dynamical motifs will help to identify new substrates and to elucidate the physiological functions of the protease in the brain and other organs. This work is part of a collaborative research program (https://www.i-proteolysis.de/). 

Speaker
Biography:

Andrei A. Korostelev is passionate about mechanisms of translation regulation. He received Ph.D. in Michael S. Chapman laboratory at Florida State University in 2003 and performed postdoctoral studies with Harry F. Noller in 2004-2010. The Korostelev laboratory at the RNA Therapeutics Institute uses recent advances in biochemical and structural methods to elucidate detailed mechanisms that govern translation and regulation of translation under stress conditions or during disease. Recent work revealed high-resolution “frames” of the motions that the translational machinery undergoes during bacterial stress responses (including the stringent response) and viral infection, as summarized on the laboratory web site: http://labs.umassmed.edu/korostelevlab/research.htm

Abstract:

Virus propagation depends on efficient synthesis of viral proteins by the host translational machinery. Internal ribosome entry sites (IRESs) of viral mRNAs mediate cap-independent initiation. Intergenic-region (IGR) IRESs of Dicistroviridae family, which includes the Taura syndrome virus (TSV) and Cricket paralysis virus (CrPV), use the most streamlined mechanism of initiation, independent of initiation factors and initiator tRNA. A tRNA-mRNA like pseudoknot of IGR IRESs binds the ribosomal A (aminoacyl-tRNA) site of the 80S ribosome (Fernandez et al., 2014; Koh et al., 2014). The pseudoknot has translocate to the P site to allow binding of the first tRNA and initiate translation.

Using electron cryo-microscopy of a single specimen, we resolved five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics (animation).

The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. 

Speaker
Biography:

Shuanghong Huo received her Ph.D. in Computational Chemistry from Boston University. She had postdoctoral training at UC-San Francisco. She is a Professor of Chemistry and Biochemistry at Clark University, Worcester, USA. Her research interest is protein folding, misfolding, and aggregation. Recently her group is developing dimensionality reduction methods and graph representations of protein free energy landscapes. 

Abstract:

In the study of protein thermodynamics and kinetics it is of paramount importance to characterize protein free energy landscapes. Dimensionality reduction is a valuable tool to complete the task. We have evaluated several methods of dimensionality reduction, including linear and non-linear methods, such as principal component analysis, Isomap, locally linear embedding, and diffusion maps. A series of criteria was used to assess different aspects of the embedding qualities. Our results have shown that there is no clear winner in all aspects of the evaluation and each dimensionality-reduction method has its limitations in a certain aspect. The linear method, principal component analysis, is not worse than the nonlinear ones in some aspects for our peptide system. We have also developed a mathematical formulation to demonstrate that an explicit Euclidean-based representation of protein conformation space and the local distance metric associated to it improve the quality of dimensionality reduction. For a certain sense, clustering protein conformations into macro-clusters to build a Markov state model is also an approach of dimensionality. We have tested inherent structure and geometric metric for state space discretization and demonstrated that the macro-clusters based on inherent structure give a meaningful state space discretization in terms of conformational features and kinetics.

Speaker
Biography:

Head, Laboratory of Biological Physics, Institute of Physics, Polish Academy of Sciences in Warsaw, Poland. Education - M.S., Department of Physics, University of Warsaw, 1973; Ph. D., Department of Physics, University of Pittsburgh, 1977; D.Sc., Department of Physics, University of Warsaw, 1984. Professorial title, 1994. Fields of interest – condensed matter theory (spin waves, spin glasses, porous media, growth processes, atomic friction, river networks, nanofluidics, self-organized nanostructures) and biological physics (large conformational changes of biomolecules within coarse-grained models, especially as induced by stretching,  proteins with knots and slipknots, protein folding, dynamics of virus capsids and other multi-proteinic structures such as a cellulosome, interaction of proteins with solids, proteins at air-water interface, modeling of proteasomes,  inference of genetic networks from the microarray data). Co-author of textbook “Theory of Quanta”, Oxford University Press 1992. 250 research papers.

Abstract:

We review the physics of  processes involving large conformational transformations in knotted proteins in bulk water and then consider folding in ribosomes and unfolding in proteasomes.  Formation of a knot is demonstrated to be facilitated by the nascent conditions at the ribosome. Knots in proteins have been proposed to resist proteasomal degradation. Ample evidence associates proteasomal degradation with neurodegeneration. One interesting possibility is that indeed knotted conformers stall this machinery leading to toxicity. However, although the proteasome is known to unfold mechanically its substrates, at present there are no experimental methods to emulate this particular traction geometry. Here, we consider several dynamical models of the proteasome in which the complex is represented by an effective potential with an added pulling force. This force is meant to induce translocation of a protein or a polypeptide into the catalytic chamber. The force is either constant or applied periodically. The translocated proteins are modelled in a coarse-grained fashion. We do comparative analysis of several knotted globular proteins and the transiently knotted polyglutamine tracts of length 60 alone and fused in exon 1 of the huntingtin protein. Huntingtin is associated with Huntington disease, a well-known genetically-determined neurodegenerative disease. We show that the presence of a knot hinders and sometimes  even jams translocation. We demonstrate that the probability to do so depends on the protein, the model of the proteasome, the magnitude of the pulling force, and the choice of the pulled terminus. In any case, the net effect would be a hindrance in the proteasomal degradation process in the cell. This would then yield toxicity via two different mechanisms: one through toxic monomers compromising degradation and another by the formation of toxic oligomers

Speaker
Biography:

Andreas Kuhn has his expertise in protein folding of membrane proteins. Studies include reconstituted systems with bacterial translocases and insertase, as well in vivo studies with Escherichia coli. For biophysical experiments the membrane proteins are purified and their folding is monitored spectroscopically in real time after their addition to liposomes. Andreas Kuhn obtained his PhD from the Universities Basel and Freiburg i. Br. 1982. After a postdoc at UCLA with Bill Wickner he continued at the Biozentrum Basel from 1986 to 1989 and accepted a professorship at the University Karlsruhe. Since 1996 he is at the University of Hohenheim in Stuttgart.

Abstract:

Most membrane proteins are inserted co-translationally by the Sec-translocase or the YidC/Oxa1/Alb3 insertases. The folding of these proteins occurs within the membrane during the interaction with the insertases. We have purified and reconstituted YidC, the membrane insertase of Escherichia coli. The protein spans the membrane 6 times, and the recently solved structure shows a hydrophilic cavity and a greasy slide between the transmembrane segments TM3 and TM5. Hydrophobic residues of TM3 and TM5 interact with the substrate, in particular with a prospective transmembrane segment of an inserting membrane protein as we documented by disulfide crossinking experiments.

The membrane insertion process can be studied with the reconstituted vesicle system. The purified substrate proteins are solubilized in 10% isopranol or kept unfolded with urea or GuHCl. When the substrate proteins are added to the proteoliposomes by dilution 1:100, they rapidly bind to YidC and become membrane inserted within 2 msec. FRET-based kinetic measurements show that the substrate proteins approach YidC to a close distance during the insertion event. Time-resolved fluorenscence anisotropy shows that the periplasmic domain of YidC moves when a substrate protein was added. This suggests that both the insertase and the substrate protein undergo conformational motions.

Speaker
Biography:

Liliane Mouawad was always interested in understanding the mechanism of action of proteins or protein assemblies. This understanding may be based on either molecular simulations or on experiments like NMR. But her expertise is primarily in molecular dynamics simulations and more precisely in normal mode analysis (NMA). She has developed several methods going from the calculation of normal modes of very large systems or of images, to the calculation of the pathway between two protein conformations, to the prediction of the compactness of a calcium-binding protein. Recently she was also involved in docking and virtual screening themes, where she has acquired enough expertise to develop a new consensus methodology to overcome some issues observed in these approaches. 

Abstract:

A microtubule is a dynamic system formed of ab-tubulins. The presence of nonhydrolyzable guanosine-5’-triphosphate (GTP)/guanosine diphosphate (GDP) on the b-tubulins provokes microtubule polymerization/depolymerization. Despite the large number of experimental studies of this dynamical process, its mechanism is still unclear. To provide insights into this mechanism, we studied the first depolymerization steps of GDP/GTP-bound microtubules by normal-mode analysis with the all-atom model. We also constructed a depolymerizing microtubule and compared it to cryo-electron microscopy tomograms (cyro-ET). The results show that during depolymerization, the protofilaments not only curve but twist to weaken their lateral interactions. These interactions are stabilized by GTP, but not evenly. Not all of the interface residues are of equal importance: five of them, belonging to the H2-S3 loop, play a special role; acting as a lock whose key is the g-phosphate of GTP. Sequence alignments of several tubulins confirm the importance of these residues.

Speaker
Biography:

Chantal Prévost is researcher at the Theoretical Biochemistry Laboratory (LBT) of the French National Research Center (CNRS), in Paris. She has developed a large expertise in studying macromolecular self-assembliy in silico, either by elaborating new algorithms for flexible proteins docking or by studying fundamental biological processes involving the transition between instable conformational substates. She presently applies this expertise to exploring the architecture or oligomeric assemblies as well as elucidating the mechanism of homologous recombination, in collaboration with experimental partners.

Abstract:

Statement of the Problem: Many proteins present highly flexible or disordered fragments, either terminal tails or surface loops. Although they often form instable and transient interactions, these fragments play essential roles in regulating macromolecular association or controlling the architecture of supramolecular complexes. The role of their conformational variability in complex formation is poorly understood and requires the development of specific approaches.

Methodology & Theoretical Orientation: We have studied the effect of protein segment conformational variability in protein-protein complex formation as well as peptide docking using theoretical docking approaches. Notably, we have developed a flexible docking method that accounts for the presence of flexible loops, together with analysis protocols that capture the entropic effects associated to structural variability in flexible docking results. 

Findings:  Whether the flexible segment is a loop or a peptide, we have found that a given mode of association can be stabilized by different  conformations of the segment.  Alternatively, different loop conformations can stabilize different modes of protein-protein association.

Conclusion & Significance: Tolerance of a binding site to conformational variability, as observed in protein-peptide docking but also in the association of proteins with flexible loops or segments, can play a role in adding a conformational entropy component to the energy of association, thus favoring the initial binding of the flexible fragment to its binding site. For proteins that associate using different binding geometries, either with different partners or along a functional pathway, loop flexibility can also be used to regulate the choice of  the binding geometry. 

Speaker
Biography:

Ramakrishna Vadrevu received his Masters degree in Physical Chemistry and his Ph.D. degree in Biophysical Chemistry.  He spent few years at the Pennyslvania State University and later at University of Massachusetts Medical School as a post doctoral fellow with Prof. C. Robert Mathews. Since 2008 he has been a faculty at the Birla Institute of Technology and Science-Pilani, Hyderabad Campus in the department of Biological Sciences.

His research focuses on understanding the role cellular environment on protein stability and folding. His research interests also include protein design and engineering, amyloid material and its applications. 

 

Abstract:

Statement of the Problem: The acquired complex three-dimensional structure of proteins is a culmination of simple structural fragments like a-a, b-b, a-b and  b-a units.  Thus, tertiary structures can be seen as a combination of basic building block motifs implying that all complex protein structures have evolved from the assembly of small independently folding super secondary structures. The TIM barrel proteins are made up of a regular repeating bab motif resulting in the strands and helices in an alternating repetitive pattern. Experimental and theoretical studies have revealed that bab unit acts as a minimal unit of stability. The success of designing super secondary motifs that fold in isolation underscores the prospects of designing and or identification of independently folding motifs from the existing protein structures. However, intriguingly, naturally occurring bab sequences from proteins that fold independently have not been identified. In our attempts we addressed the finding of ‘needles in hay stick’ scenario by an exhaustive sequence and structural space search of the bab units                                                                                       from the TIM barrels.   Methodology & Theoretical Orientation: The search approach implemented in this work considered features such as alpha helical propensity, loop length, loop dynamics, residue preferences in loops, long range side chain main chain interactions etc., to shortlist bab units with strong propensity to fold in isolation.  The prospective bab candidates thus shortlisted from the TIM barrels have been further subjected to structure forming tendency employing a combination of Monte Carlo and Molecular dynamics simulations to assess their foldability and stability. Conclusion & Significance: The prediction of some independently folding bab candidates from TIMs are enabling us to experimentally asses their folding and stability. The identification and analysis of independently folding bab units that exist naturally will not only provide substantial information on nature’s design strategies and evolution of protein conformations but also help to design/engineer novel proteins. 

  • Track 6: Frontiers in Structural Biology | Track 7 : Structural Molecular Biology
Biography:

Petra Fromme received her masters at the Free University in Berlin in Biochemistry (1985) and then received her doctorate in Chemistry at the Technical University in Berlin (1988) where she then became a professor in 1992.  During this time she developed and pursued her fascination with understanding the function of membrane proteins by investigating and determining their atomic structures.  In 2002, Dr. Fromme joined Arizona State University as a professor of Chemistry and Biochemsitry where she has worked with distinguished colleagues from around the world to pioneer a new technique for imaging proteins using extraordinarily powerful x-ray lasers that has the capability to make movies of these fascinating proteins in action.

Abstract:

Biomolecules are highly dynamic; however, most structures only provide a static picture of the molecule. Serial Femtosecond Crystallography (SFX) provides a novel concept for structure determination, where X-ray diffraction “snapshots” are collected from a fully hydrated stream of nanocrystals, using femtosecond pulses from high energy X-ray free-electron lasers (XFELs), where diffraction is observed before destruction takes effect [1]. The first proof of concept of serial femtosecond crystallography was achieved using Photosystem I, a larger membrane protein complex involved in Photosynthesis as a model system [2]. The structure of non-damaged biomolecules can now be determined, unraveling their function at the atomic scale [3],[4] that include important human membrane-bound receptors [6], [8] [9]. SFX opens a new avenue for determination of protein dynamics, with the goal of molecular movies of biomolecules “in action”.  First experiments on the proof of principle for time resolved serial femtosecond nanocrystallography have been performed on proteins in Photosynthesis, where first snapshots of steps in water splitting reaction have been observed [10]. A new concept based on continuous X-ray diffraction extends resolution beyond Bragg diffraction and allows for direct phasing of X-ray diffraction data [11]. TR-SFX studies extend to atomic resolution where the first steps in photosensing were recently revealed at a time scale of femtoseconds using the photoactive yellow protein [12,13]. This pioneering work paves the way for the determination of molecular movies of the dynamics of membrane proteins "at work" in the future.

The talk will close with a progress report on the development of compact femto and attosecond X-ray Sources at DESY (AXSIS) and at ASU (CXLS and CXFEL) , which will provide unique new opportunities to study the ultrafast dynamics of reactions in photosynthesis with a combination of X-ray diffraction, X-ray spectroscopy and ultrafast optical spectroscopy [14].

Biography:

Kurt Ballmer-Hofer focused his research at PSI on the structural and functional analysis of receptor tyrosine kinases, in particular on Vascular Endothelial Growth Factor Receptors, VEGFRs. In collaboration with partner labs his team solved the structures of VEGF ligands, the ligand binding domain of VEGFR-2, and -3, and of the full-length extracellular domain of VEGFR-1 in complex with VEGF. The data of these studies led to the discovery of allosteric receptor regulatory sites in subdomains 4, 5 and 7. Antibodies and DARPins specifically binding to these domains showed strong inhibition of receptor activation and downstream signaling both in vitro and in vivo in angiogenesis model systems. 

Abstract:

Vascular Endothelial Growth Factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (RTKs), VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single particle electron microscopy, small angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and the spatial arrangement of individual receptor subdomains. Here we describe the structure of the full-length VEGFR-1 extracellular domain (ECD) in complex with VEGF-A at 4 Å resolution. We combined X-ray crystallography, single particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in Ig-domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts for receptor activation and identify them as allosteric regulatory sites of VEGFR-1.  

Based on our structural data we also investigated the function of Ig-domains 4, 5 and 7 in VEGFR-2, the primary receptor driving angiogenesis and vasculogenesis in response to VEGF administration. The basic domain structure of VEGFR-2 is very similar to VEGFR-1, the ECD of both receptors consists of 7 Ig-domains, D1-D7. Mutagenesis studies based on the VEGFR-1structure confirmed that Ig-domains 4 and 7 fulfill an essential regulatory function in receptor activation and may thus represent putative targets for pharmacological intervention. We isolated highly specific antibodies and DARPins (Designed Ankyrin Repeat Proteins) specific for domains 4 or 7. A subset of these reagents efficiently blocked receptor activation and inhibited VEGF-dependent signaling in vitro in endothelial cell cultures. Most importantly, a domain 4-specific DARPin efficiently blocked vessel development also in vivo in a mouse angiogenesis model. In this model endothelial cell spheroids were implanted in matrigel into mice, and cell growth and vessel formation were monitored in the absence and presence of inhibitor. Our study thus revealed a novel approach for therapeutic targeting of aberrant blood vessel development.  

Speaker
Biography:

Jeffrey Urbauer earned bachelor’s and doctoral degrees in chemistry from the University of Nebraska-Lincoln. He pursued postdoctoral studies at the University of Wisconsin-Madison as an NIH postdoctoral fellow and at the University of Illinois Urbana-Champaign. He held faculty appointments at the State University of New York at Buffalo, the University of Pennsylvania, and the University of Kansas before joining the faculty in the Department of Chemistry and the Department of Biochemistry and Molecular Biology at the University of Georgia. At the University of Kansas the Mortar Board National College Senior Honor Society awarded him with the Outstanding Educator Award. His research interests include structural biology, protein biophysics and NMR spectroscopy.

Abstract:

Statement of the Problem: Oxidation of methionine residues in proteins to methionine sulfoxide is a prevalent, reversible post-translational modification. Changes in protein structure and function accompany oxidation due to polarity and steric differences between methionine and the sulfoxide. We are investigating the consequences of methionine oxidation in the regulatory protein calmodulin (CaM), a key      calcium signal transducer with nine methionine residues, in hydrophobic pockets of its opposing globular domains, which interact with target proteins. CaM with oxidized methionine residues accumulates under conditions of oxidative stress, and because of its central role in biology, it is important to understand the functional effects of these alterations and their physical origins.  Methodology: Methionine residues in CaM are easily oxidized in vitro with hydrogen peroxide. To study the effects of oxidation of specific methionine residues, leucine was substituted for methionine at remaining sites. A combination of functional assays, single molecule studies, and NMR spectroscopy were used to assess functional and structural consequences of methionine oxidation.  Findings: For the best studied case, activation of the plasma membrane Ca-ATPase (PMCA) by CaM, impaired CaM function is due to oxidation of a single C-terminal methionine. Single molecule experiments indicate non-productive binding of oxidized CaM to the PMCA. High resolution NMR studies demonstrate significant structural perturbation in the C-terminal globular domain of oxidized CaM and an inability to anchor the PMCA to this domain. Conclusion & Significance: The functional effects of methionine oxidation in CaM are highly target dependent, as is the degree to which selective oxidation of particular methionine residues in CaM affects function. The results of CaM activation of the PMCA also indicate that both high-affinity productive and non-productive complexes of oxidized CaM with targets are possible. These facts indicate that a comprehensive understanding of the metabolic consequences of CaM oxidation will be challenging.

Speaker
Biography:

I received by BSc from Texas A&M University in 1997.  I began crystallography in the laboratory of Yousif Shamoo at Rice University.  During my graduate studies I worked on the structural mechanism of the human sliding clamp and its interactions with DNA replication proteins.  I received my PhD in 2005.  I completed 2 successful post-docs.  The first was at the Scripps Research Institute from 2005-2007 working on structural studies of nuclear receptors including PPAR, RXR, ER, and TR.  My second post-doc was with Jim Sacchettini in the Houston Medical centre and I was a part of the TB structural genomics consortium.  I received my first faculty position at the University of Adelaide in 2012 as a lecturer.  I was tenured in 2015 and promoted to Senior Lecturer in 2016.  Do to my continued collaboration with Scripps, I was also appointed adjunct professor of the Scripps Research Institute in 2016.

Abstract:

Synthetic full agonists of PPARγ have been prescribed for the treatment of diabetes due to their ability to regulate glucose homeostasis and insulin sensitization. While the use of full agonists of PPARγ has been hampered due to severe side effects, partial agonists and antagonists have shown promise due to their decreased incidence of such side effects in preclinical models. No kinetic information has been forthcoming in regard to the mechanism of full versus partial agonism of PPARγ to date and little structural and dynamic information is available which can shed light on the mechanistic difference between full and partial agonists as well as antagonists. We have used X-ray crystallography, cellular assays, Hydrogen Deuterium Exchange (HDX), and Surface Plasmon Resonance (SPR) to probe the mechanism of several PPARγ partial agonists and antagonists. Our findings demonstrate that not only do partial agonists and antagonists act through distinct transcriptional mechanisms, they also demonstrate differences in structure, dynamics, and kinetics as compared to full agonists.

Speaker
Biography:

Maria Bykhovskaia is an expert in synaptic transmission. Her lab combines molecular modeling and computations with electrophysiology, microscopy, and molecular biology approaches. She hold a Professor’s position in the WSU. Her Ph.D. training was in protein molecular modeling, and subsequently she used a postdoc in computational neuroscience to initiate a career devoted to the study of presynaptic mechanisms and plasticity. As a PI, she has developed in her lab expertise in electrophysiology, live confocal imaging, and electron microscopy. The lab combines these experimental approaches with mathematical modeling to understand the fundamental mechanisms of release of neuronal transmitters.

Abstract:

Neuronal transmitters are released via the fusion of synaptic vesicles with the plasma membrane. Vesicles dock to the membrane via a protein complex termed SNARE, which contains membrane attached (t-SNARE) and vesicle attached (v-SNARE) proteins. The fusion occurs in response to a calcium inflow, and the vesicle protein Synaptotagmin (Syt) serves as a calcium sensor. A cytosolic protein Complexin (Cpx) interacts with the SNARE complex, restricting the spontaneous fusion. Although molecular interactions of these proteins have been extensively studied, it is still debated how Syt dynamically interacts with the SNARE protein complex, Cpx, and lipid bilayers to trigger lipid merging. To elucidate these mechanisms, we combined molecular dynamics (MD) simulations with molecular biology and genetic approaches in Drosophila. Basing on MD simulations, we created a model of the protein fusion machinery wherein Cpx dynamically interacts with v-SNARE, preventing full SNARE assembly. Our MD simulations also elucidated how Syt interacts with lipid bilayers, causing lipid bulging that may precede the formation of the stalk and the fusion pore opening. Finally, our simulations predicted direct interactions of Syt with the SNARE-bound Cpx. The developed molecular model enabled us to predict new mutations in v-SNARE and Cpx that alter the fusion process. To test these predictions, we generated Drosophila lines with single point mutations and investigated how these mutations affect  the kinetics of transmitter release. The results of these experiments suggest that our model creates the basis for systematic approach to manipulating the fusion machinery based on theoretical predictions derived from MD simulations.

Speaker
Biography:

Mi Sun Jin - Assistant Professor, 2014-Present, School of Life Sciences, GIST;

Education - B.S. 2002, Sogang University; M.S. 2004, KAIST; Ph.D. 2008, KAIST (advisor: Jie-Oh Lee)

Professional Experience - Postdoctoral Fellow, 2008-2009, KAIST (advisor: Jie-Oh Lee),
                                             Postdoctoral Fellow, 2009-2013, Purdue University (advisor: Jue Chen),
                                             Research Specialist, 2013-2014, Purdue University

Abstract:

Speaker
Biography:

Senyon Teddy Choe, is a Professor of Biology and the founding director of Drug Discovery Collaboratory at UCSD. After his Ph.D study in Biophysics and Medical Physics at Univ. California, Berkeley. He joined the Salk Institute in 1993 as the founding faculty member of the Institute’s new Structural Biology Laboratory, and remained so through 2015. His research group has focused on understanding how cells talk to each other. An extension of these works explores designing synthetic biologics to modulate stem cells and sick cells directly. His major honors include election in 1999 to the Fellow of American Association for the Advancement of Science. He recently founded joint Center for Biosciences to translate discovery to medical applications to focus on protein engineering and developing new stem cell therapy. He currently leads Mogam Institute for Biomedical Research in Korea aiming for biologics discovery in the areas of infectious diseases and cancer.

Abstract:

Discovery of new biologics presents new challenges and opportunities and revenues for biologics are rapidly growing in biopharmaceutical industry. We exploited our detailed structural knowledge of three-dimensional structures of BMPs, their receptors, and their antagonists to engineer synthetic BMP ligands. By use of a novel protein engineering strategy that we termed RASCH (Random Assembly of Segmental Chimera and Heteromers), we have set out to design a synthetic biologics (synbiologics®) for bone and cartilage therapy. In the case of bone fusion, we have used Activin and BMP-2, which are the members of TGF-beta superfamily, to create AB204 (Allendorph et al., 2011). AB204 is a synthetic 50:50 chimera of the two ligands, which indeed shows highly effective bone-forming capability (Yoon et al., 2014). In the case of cartilage, we have used Activin and BMP-6 to create AB604, again a 50:50 chimera of the two. AB604 shows all the properties of super BMP6, surpassing the functional characteristics of natural BMP6.

This approach promises to be a very powerful way to harness different biological functionalities of natural ligands to merge into one synthetic designer molecule such that it goes beyond what Mother Nature could provide a new means to meet various unmet clinical needs.

  • Special Session on Structural Biology of Biomembranes
  • Track 8: Drug Designing

Session Introduction

Dirksen Bussiere

Novartis, USA

Title: TBA
Speaker
Biography:

Dirksen Bussiere Led 22-FTE structural biology and biophysics research effort (X-ray crystallography, biophysics, NMR and protein biochemistry support) on multiple structure-based projects encompassing variety of anti-infective, metabolic, and oncology targets

Led fragment-based screening and biophysics team which spans four functional areas (Structural and Biophysical Chemistry, Computational Chemistry, Biochemical Lead Discovery, and Protein Sciences)

Led multiple hit-finding project teams focused on finding hits for anti-infective and oncology
targets

Served as lead structural biologist. biochemist and biophysicist on multiple oncology and anti-infective drug discovery project teams

Provided molecular modeling, drug design, and bioinformatics support to project teams

Provided fragment-based screening support for project teams, including NMR and biophysical screening

Named Novartis Leading Scientist in 2007, an award & title given to less than 1% of Novartis scientists world-wide

Abstract:

To be updated

Speaker
Biography:

Michael Hennig is a drug discovery research manager with 22 years of experience in pharmaceutical industry. He co-founded and is CEO and Chairman of the board of leadXpro, an emerging biotech company and spin-out of the Paul Scherrer Institute (ETH, Switzerland) that is dedicated to structure based drug discovery of membrane protein targets. Formerly he worked 20 years at Roche research Basel, Global Head and Principle Leader of discovery technologies with responsibility for structure based drug discovery, protein science, assay development and HTS, corporate compound library, stem cell platform. In addition, he is guest Professor at the University of Basel in structural biology, gives lecture series in pharmacy, is author of  >75 paper and lecturer at conferences, inventor of 8 patents in areas of technology, discovery and formulation of drug substances.

Abstract:

Today, structure based drug discovery is well implemented in the drug discovery engine of many pharmaceutical companies. Whereas soluble proteins are managed well within the project timelines and portfolio changes in pharmaceutical industry, transmembrane proteins still represent a significant challenge. LeadXpro combines expertise of drug discovery, excellence in high quality solubilized and purified membrane protein science and use of cutting edge biophysical methods like X-ray data collection at synchrotron and FEL sources, single particle cryo-electron microscopy, SPR and others. Strong relationship between leadXpro and Swiss large research facilities like PSI-SLS and SwissFEL as well as C-CINA will enable advances in structure determination of challenging membrane protein drug targets that have not been feasible before. Knowledge of the drug candidate and protein target 3d-structure, together with the full characterization of its interaction by biophysical binding and functional assays will enable to generate novel and better lead molecules for future medicines.

Examples of recent developments include the successful fragment screening for the GPCR neutrotensin receptor 1, a fragment screening with 6369 compounds was performed with SPR and 44 hits identified. Finally, 4 selected hits were validated in NMR experiments and computational analysis gave insight into the potential fragment-binding location and interactions to inspire further chemistry efforts. Furthermore, serial crystallography performed at synchrotron and free electron laser enables structure determination on challenging drug targets. Advantages are analysis at physiologically more relevant room temperature (no freezing of crystals required), low or no radiation damage and the use of very small crystals.