Scientific Program

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

Day 2 :

Keynote Forum

Timothy A. Cross

Florida State Univ. & National High Magnetic Field Lab, USA

Keynote: Membrane Protein Structure, Dynamics & Function: Oriented Sample and Magic Angle Sample Spinning Solid State NMR
Structural Biology 2017 International Conference Keynote Speaker Timothy A. Cross photo
Biography:

Timothy Cross has more that 30 years of experience characterizing membrane proteins in lipid bilayer environments using solid state NMR of liquid crystalline bilayer preparations of peptides and proteins. This has brought to light a fundamental understanding of membrane protein biophysics that has led to detailed functional characterizations of membrane channels – in particular the gramicidin monovalent cation channel and the influenza A M2 proton channel. In both systems the unique features of the membrane environment play crucial roles in the functional mechanisms and kinetics of ion conductance. Is this understanding of the influence of membrane and lipid environments through the use of solid state NMR that has driven his research at the frontier of membrane protein biophysics.

Abstract:

Statement of the Problem: Unlike water soluble proteins that have a relatively homogeneous environment, membrane proteins exist in a dramatically heterogeneous environment. The result is protein structure that is stabilized by a different balance of molecular interactions for the membrane embedded portion of the protein compared to the water soluble or membrane interfacial regions of the protein. The result is a need to model the membrane environment as closely as possible to that of the native environment for structural, dynamic and functional characterizations. Methodology: Biological solid state NMR provides a unique opportunity to model the membrane environment with liquid crystalline lipid bilayers and a wide variety of lipids. The samples can be prepared either as liposomes for magic angle sample spinning (MAS) or as uniformly oriented samples (OS) for the spectroscopy. The former provides solution like spectra for both distance and isotropic chemical shift restraints, while oriented samples provide absolute restraints that restrain the atomic sites in the protein structure to the bilayer normal. In addition to structural restraints it is possible to characterize the protein’s dynamics and kinetic rates. Findings, Conclusion & Significance: The structure, dynamics and kinetics associated with the M2 proton channel from influenza A have been characterized yielding a unique mechanism for proton transport by this important drug target. In addition, the cholesterol binding to M2 has been found to stabilize the amphipathic helix in the lipid interface an essential feature for this protein’s functional role in viral budding. Recent structural studies of the CrgA protein from Mycobacterium tuberculosis  have characterized a dimeric structure stabilized primarily by intermolecular b-sheet in the membrane interfacial region. The protein is part of the cell division apparatus and appears to play a role in recruiting multiple proteins to the divisome, potentially through its transmembrane domain.

Keynote Forum

Yuri L. Lyubchenko

University of Nebraska Medical Center, USA

Keynote: Protein-protein interaction and amyloid cascade hypothesis for Alzheimer’s disease
Structural Biology 2017 International Conference Keynote Speaker Yuri L. Lyubchenko photo
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:

The amyloid cascade hypothesis is currently considered as the main model for a vast number of neurodegenerative diseases including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Numerous studies have shown that amyloidogenic proteins are capable of spontaneous assembly into aggregates, and eventually form fibrillar structures found in amyloid or amyloid‐like deposits. However, there is a serious complication with translating current knowledge on amyloid aggregation in vitro to understand the aggregation process in vivo. If the critical concentration for the spontaneous aggregation of Aβ peptide in vitro is in the micromolar range, physiological concentrations of Aβ are in the low nanomolar range making impossible amyloids to assemble. We have discovered a novel on-surface aggregation pathway that allows for spontaneous assembly of amyloid beta peptides at the physiological concentration range. Our combined experimental and computer modeling approaches demonstrate that the on-surface aggregation is a dynamic process, so the assembled aggregate can dissociate from the surface to the bulk solution. As a result, the dissociated oligomers can play roles of seeds for aggregation in the bulk solution, or start a neurotoxic effect such as phosphorylation of the tau protein to initiate its misfolding and aggregation. Both processes lead to neurodegeneration. Importantly, in the vast majority of cases, we found that aggregates formed on the surface are oligomers, which are considered to be the most neurotoxic amyloid aggregates. Therefore, we posit that on-surface aggregation is the mechanism by which neurotoxic amyloid aggregates are produced under physiological conditions. A change in membrane properties leading to an increase in affinity of amyloid proteins to the membrane surface facilitates the assembly of stable oligomers. The proposed model is a significant departure from the current model as it directs the development of treatments and preventions towards approaches that control the cell membranes properties and composition preventing the on-surface aggregation process.   

  • Track 7: Structural Biology in Complexity Arenas and Cancer Research
Biography:

Charlie Carter is an X-ray crystallographer who studies the origin, evolution, and structural biology of aminoacyl-tRNA synthetases. His research group introduced the use of Urzymes—highly conserved structural cores that retain large fractions of the transition-state stabilization free enregies of full length enzymes—as experimental models of ancestral enzymes. 

Abstract:

Enzyme mechanisms, especially those that couple NTP hydrolysis to mechanical work and information, use sophisticated dynamic networks to transduce active-site chemistry into domain motions that change binding affinities [1]. We measured and cross-validated the energetics of such networks in B. stearothermophilus Tryptophanyl-tRNA synthetase (TrpRS) using both multi-mutant and modular  thermodynamic cycles [2]. Coordinated domain motions develop shear in a core packing motif conserved in >125 different protein superfamilies [2]. Multi-dimensional combinatorial mutagenesis showed that four side chains from this “molecular switch” move coordinately with the active-site Mg2+ ion in the transition state for amino acid activation [3]. A modular thermodynamic cycle consisting of full-length TrpRS, the Urzyme, and the Urzyme plus each of the two domains deleted in the Urzyme [4]gives similar energetics. These complementary experiments establish that catalysis and specificity in full-length TrpRS are both coupled by 5 kcal/mole to:  (i) the core packing region where domain movement generates shear, and (ii) the simultaneous motion of the two domains relative to the Urzyme. Theory shows that the minimum action path algorithm estimates thermodynamically meaningful contributions of domain movement to kinetic rates [5]. Correlations between those parameters, the experimental rates, and structural variations induced in the combinatorial mutants confirm that these estimates are realistic. These results validate our previous conclusion that catalysis by Mg2+ ion is coupled to the overall domain motion (3). Computational free energy surfaces demonstrate that TrpRS catalytic domain motion itself is endergonic but is driven thermodynamically by PPi release [5,6]. Comparison of the impact of combinatorial mutagenesis on pre-steady state and steady-state rates confirm that dynamic active-site pre-organization endows TrpRS with the elusive conditionality of NTP utilization on domain motion.           

Biography:

Tzu-Ching has completed his PhD from University of Nebraska Medical Center in 1999 and postdoctoral studies from Cold Spring Harbor Laboratory in 2003. Since then, he has been working at Academia Sinica, the premier government-funded institution in Taiwan. He is now a Research Fellow with professorship jointly appointed by National Taiwan University. He has published more than 40 papers in reputed journals and has been serving as an advisory board member of competitive journals.

Abstract:

The Ras signaling cascade acts as a key driver in human colon cancer progression. Among the modules in this pathway, p38gamma (MAPK12) and its specific protein tyrosine phosphatase PTPN3 (PTPH1) are critical regulators responsible for Ras oncogenic activity. However, the molecular basis for their interaction is completely unknown. Here we report the unique architecture of the PTPN3-p38gamma complex by employing an advanced hybrid method integrating X-ray crystallography, small-angle X-ray scattering (SAXS) and chemical cross-linking/mass spectrometry (CX-MS). Our crystal structure of PTPN3 in complex with the p38gamma phosphopeptide presented a unique feature of the E-loop that defines the substrate specificity of PTPN3 towards fully activated p38gamma. The low-resolution structure demonstrated the formation of an active-state or a resting-state complex of PTPN3-p38gamma. We showed a regulatory function of PTPN3’s PDZ domain, which stabilizes the active-state complex through interaction with the PDZ-binding motif of p38gamma. Using SAXS and CX-MS approaches, we found that binding of the PDZ domain to the PDZ-binding motif lifts the atypical auto-inhibitory constraint of PTPN3, enabling efficient tyrosine dephosphorylation of p38gamma to occur. Our findings emphasize the potential of structural approach for PTPN3-p38gamma complex that may deliver new therapeutic strategies against Ras-mediated oncogenesis in colon cancer.

Federica Cossu

Institute of Biophysics at the National Research Council (IBF-CNR), Italy

Title: Type I BIR domain inhibitors in cancer therapy: designing drugs to modulate the NF-κB pathway
Biography:

Federica Cossu has always been interested in the field of cancer research, being fascinated by structural studies of crucial macromolecules and protein complexes involved in the cellular processes of cell death/survival. She gathered experience in cloning, expression, purification and crystallization of recombinant proteins, mainly belonging to the field of cancer. During the last years, she focused on the structure-based design of small molecules to be developed as drug candidates directed to pre-clinical studies. The success of this activity is proven by one patent, one award for her PhD thesis and several publications in the field. She progressively improved her knowledge on biophysical techniques for the study of proteins and on the in silico analysis of protein structures. She collected experiences in European laboratories, including several short visits/experiments at the ESRF synchrotron in Grenoble and at SOLEIL in Paris. She has been working in lab equipe for ten years, covering different positions within the research group, from master degree student to post-doc. She has been the supervisor of students, also giving lessons on the structural approaches applied to cancer therapy.

Abstract:

Inhibitors of apoptosis proteins (IAPs) constitute a family of conserved proteins whose over-expression enhances cell survival and resistance to anticancer agents. IAPs are E3 ligases, ubiquitylating substrates for the regulation of NF-kB; furthermore, they sequester caspases to prevent apoptosis. IAPs interactions occur through type I and type II BIR (Baculovirus IAP repeat) domains. Smac-mimetics (SM) mimicking the active N-terminal peptide of Smac-DIABLO, the natural antagonist of IAPs, have been shown to sensitize cancer cells to apoptosis. SM interact with type II BIR domains of IAPs, thus relieving caspases from X-linked IAP (XIAP) inhibitory activity and leading to cellular IAPs (cIAPs) auto-ubiquitylation and proteasomal degradation within minutes from exposure. Although SM are currently promising candidates for cancer therapy, some cancer cell lines present SM-resistance due to renewed cIAP2 activity and re-activation of NF-κB. IAPs-mediated regulation of NF-kB signaling is based on the formation of different protein-protein complexes, regulating ubiquitin-dependent signal transduction cascades. The type I BIR domain from different IAPs has been recognized as a pivotal platform for the assembly of such complexes.

 

We analyzed the surface of type I BIR domains (X- and cIAP-BIR1) to identify the hot-spots for the relevant protein-protein interactions. Virtual docking using libraries of compounds returned hits (NF023 and analogues) able to impair BIR1-based complexes with predicted low micromolar affinities that were experimentally confirmed. To this purpose, in vitro assays include fluorescence-based and biophysical techniques (Thermofluor, Microscale Thermophoresis, SEC, DLS, SLS). Crystallography on the protein-ligand complexes is the core of the structure-driven approach used for the iterative optimization of specific and selective drug candidates. Treatment of cancer cell cultures with the selected compounds will verify their effects on the modulation of IAPs-dependent signaling cascades. This represents a novel strategy to promote apoptosis in cancer and will unravel new insights on the regulation of NF-kB pathway.

Biography:

Mrinal is a Medicinal Chemist and carries thirteen years of experience in small molecule therapeutics via orthosteric/allosteric modulation of GPCR, ion channels and enzymes with a track record in delivering pre-clinical candidates for metabolic disorders, pain, oncology and anti-infective programs. He completed his MS and PhD from internationally reputed Indian Institute of Technology in Bio-organic Chemistry and was awarded Post Doctoral Fellowships in the field of Chemical Biology from University of Uppsala, Sweden, National Institute on Aging in collaboration with The Johns Hopkins University, USA and University of Basel, Switzerland. He is a coauthor
and con-inventor of 10 patents, 15 articles and 1 book chapter.

 

Abstract:

Cancer is a disease in which cells divide uncontrollably with the potential to invade or spread to other parts of the body. Over 100 cancers affect human and ~14 million new cases of cancer occurred globally in 2012 resulting ~15% of deaths. Towards our aim in improving the quality of life for the patients suffering from cancer, we are working on two approaches: 1] DNA has played a key role as molecular target in cancer therapeutics. Compounds that target DNA are some of the most effective agents in clinical use and increased in
cancer patients’ survival. Due to their severe toxicity, however, there has been considerable excitement in targeting unusual, non-canonical structures of DNA. In that regard, G-quadruplex structures have drawn interest for studying their potential involvement to inhibit cancer growth and a compound Quarfloxin has even been reached Ph2 clinical trial. 2] On the hand, protein-protein interaction [PPI] is of importance in the regulation of cellular processes and is consequently implicated in the development of various disease states including cancer. The interaction between protein molecules, through Van-der Waals force, Hbonding interaction etc., leads to the formation of a larger
protein complexes performing a specific function. Recently, it is demonstrated, certain classes of PPI can help medicinal chemists to develop inhibitors, and the first PPI inhibitor has entered in clinical development. The presentation will focus on modeling, design, synthesis of novel small molecules and their biological evaluation either as G-quadruplex stabilizing agents or PPI inhibitors. Efficiency of the synthetic compounds was performed to assess the G-quadruplex binding affinity using different biophysical studies. For the PPI inhibitors, potential cell growth inhibition was tested against different cancer cell lines followed by dose response studies. Finally, all the selected compounds were evaluated for liver microsomal stability, aqueous solubility, CYP inhibition studies for druglikeness.
 

Biography:

The Buck laboratory studies two receptor families responsible for cell guidance and positional maintenance (Plexins and Ephrins), both with key involvement in cardiovascular and neuronal development and disease, esp. cancer. We use a wide range of structural biology (solution NMR / x-ray crystallography) and protein biophysical tools (CD, fluorescence spectroscopy, ITC and SPR) in a problem oriented approach. Part of the laboratory also pursues computational modeling and molecular dynamics to provide additional perspective on the problems, provide new insights into the experimental data and to suggest further studies.  Small GTPases and their interaction with the plexin receptor cytoplasmic domains has been a major focus of the laboratory and recently we have become very interested in protein-membrane interactions; both the transmembrane regions of the receptors as well as the transient interactions of receptor and GTPase domains with membranes

Abstract:

It is now recognized that protein-protein interactions in solution are often dynamic, especially if the binding affinities are only moderately strong. Dynamics originate in part from the interconversion between structures of the protein complex, e.g. one bound state that is in equilibrium with one or several alternate configurations. We determined the structure of such a complex using NMR restraints [1] and saw the transitions between different configurations in microsecond length all-atom molecular dynamics simulations [2]. Recently, we also studied the dissociation process of mutant complexes which had a weakened primary interaction interface. Those simulations suggested that there is no single dissociation pathway, but that the separation first involves transitions to binding interfaces with fewer/weaker contacts [3].

Comparison is made between experimental NMR relaxation measurements on the ps-ns as well as µs-ms timescale with the microsecond all atom simulations, also in the context of new simulations of the protein association process. The functional significance of the protein complex alternate states and their dynamics are discussed.

In a second part of the presentation, we consider a second system involving transient interactions; this time between K-Ras and the lipid bilayer of the plasma membrane [4]. Our recent simulations the full length GTPase at different membranes reveal the underlying rules of the interactions, emphasizing electrostatic contacts but also protein topology [5].  Again, simulations are compared with NMR experiments, carried out at model systems for the membrane.

Biography:

Guyeux has a record of about 120 scholarly publications. Since 2010, he published 43 articles in peer-reviewed international journals (as a co-author, including the top-ranked ones in the areas of computer science and interdisciplinary applications, such as AIP Chaos, Plos One, or Clinical Infectious Diseases). He is co-author of 2 book chapters and 2 scientific monograms. He is also author of 4 software patents, 53 articles that appeared in proceeding of peer-reviewed international conferences. His topics of research encompasses bioinformatics, discrete dynamical systems, and information security.

Abstract:

In our first theoretical studies about folded self-avoiding walks, we have raised raises several questions regarding the protein structure prediction problem and the current ways to solve it. In one category of PSP software, the protein is supposed to be synthesized first as a straight line of amino acids, and then this line of a.a. is folded out until reaching a conformation that optimizes a given scoring function. The second category consider that, as the protein is already in the aqueous solvent, it does not wait the end of the synthesis to take its 3D conformation. So they consider SAWs whose number of steps increases until the number of amino acids of the targeted protein and, at each step, the current walk is stretched (one amino acid is added to the protein) in such a way that the pivot k  placed in the position that optimizes the scoring function. We have proven that the two sets of possible conformations are different. So these two kinds of PSP software cannot predict the same kind of conformations.

We have proven too that the folding process G in the 2D model is chaotic according to Devaney. A consequence of this theorem is that this process is highly sensitive to its initial condition. If the 2D model can accurately describe the natural process, then this theorem implies that even a minute difference on an intermediate conformation of the protein, in forces that act in the folding process, or in the position of an atom, can lead to enormous differences in its final conformation. In particular, it seems very difficult to predict, in this 2D model, the structure of a given protein by using the knowledge of the structure of similar proteins. Let us remark that the whole 3D folding process with real torsion angles is obviously more complex. And finally, that chaos refers to our incapacity to make good prediction, it does not mean that the biological process is a random one.

Biography:

Fabio C. L. Almeida has his expertise in protein structure and dynamics by nuclear magnetic resonance (NMR). He solved the structure of several proteins by NMR. He has important contribution in the structure and dynamics of plant defensins. Fabio and his group showed that despite the conserved folding, defensins display a wide variation in dynamics, which enabled the mapping of binding regions and description of the mechanism of membrane recognition. The group also showed that dynamics are also the key for the understanding of the mechanism of membrane recognition of antimicrobial peptides. Pre-existent order in flexible peptides permits discrimination between the regions of specific and non-specific binding. Fabio´s group also described the structure and dynamics of the water cavity of thioredoxin, which is an essential structural element for catalysis. Fabio is the director of the National Center of NMR (CNRMN, http://cnrmn.bioqmed.ufrj.br) and president of the Brazilian NMR Association (AUREMN).

Abstract:

Proteins are dynamic entities able to move in a wide range of timescales that goes from picoseconds to seconds. Motions that occur in microseconds to seconds define biologically relevant events that are frequently involved in binding, allostery and catalysis1,2. In our laboratory, we used relaxation parameter, relaxation dispersion experiments and molecular dynamic simulation to correlate conformational equilibrium with molecular recognition and catalysis.

Dengue and Zika are major arthropod-borne human viral disease, for which no specific treatment is available. The flavivirus capsid protein is the trigger of virus assembly. Capsid proteins are located at the cytoplasm bound to lipid droplets (LD). Binding to LDs are essential for virus assembly3,4. We previously showed that the positively charged N-terminal region of Dengue virus capsid protein prompts the interaction with negatively charged LDs, after which a conformational rearrangement enables the access of the central hydrophobic patch to the LD surface5. We also showed the participation of the intrinsically disordered region in binding and possible regulation of capsid assembly6.

We probed the structure and dynamics of Dengue virus and Zika virus capsid proteins (DENVC and ZkC) by nuclear magnetic resonance. They bind lipid droplets (LD) in the cytoplasm, which mediates virus assembly in an unknown way. We showed that the dynamics of the capsid protein is intrinsically involved in the mechanism of LD and RNA binding and virus assembly. We also measured binding to nucleic acids and probed the assembly using small angle x-ray scattering and negative staining electron microscopy. The understanding of the participation of the intrinsically disordered N-terminal region and its dynamics helped us propose a mechanism for Dengue and Zika virus assembly and to develop a peptide with the potential to block virus assembly.

ACKNOLEDGEMENTS: FAPERJ, CAPES, CNPq, INBEB-CNPq.

Speaker
Biography:

Marta obtained Ph.D. in 2007 in nanomaterials from Cranfield University. In 2011, she joined Structural Biology group led by Alaistair Lawson at UCB. For the past five years she has been developing expertise in small molecule drug discovery using a fragment based approach. She had also been involved in the assay development for a novel antibody-enable drug discovery approach. Prior to joining UCB she was awarded a Postdoctoral Fellowship at the Institute of Food Research in Norwich to study the mono and multilayer films used to encapsulate active ingredients for a controlled and site-specific delivery within the GIT. During her time at IFR she worked on numerous projects including a commercial project for Pfizer focused on AFM imaging of proteins and polysaccharides constituting vaccines. She has also been part of the team under the leadership of Claudio Nicolletii investigating the outcome of consumption of the probiotic L. casei Shirota in subjects with Seasonal Allergic Rhinitis. 

Abstract:

Protein-protein interactions (PPIs) are of critical importance in the majority of biological cellular processes including DNA repair, immune, and allergic responses. Despite their therapeutic relevance, PPIs are intrinsically challenging targets due to complexity of interactions, assay tractability and the lack of well-defined binding pockets at the interacting surfaces.  

In the quest for small molecule drug candidates targeting PPIs, there have been a number of different approaches adopted, which include: use of existing lead or drugs, natural products, high-throughput screening and more recently established powerful fragment-based drug discovery.

At UCB we have integrated the fragment-based methodology with biological and structural information obtained from antibody-validated protein targets, to develop specific small molecule inhibitors of PPIs. An ensemble of biophysical methods (i.e. SPR, ITC, FRET, MS and ligand-based NMR), corroborated by functional data, were employed to identify and validate fragment hits that constituted the starting point for our PPI inhibitor drug discovery programs. We have also employed antibodies as research tools to hold target proteins in biologically active conformations, aiding the discovery of new small molecules for challenging targets. By holding the target protein in biologically relevant conformations, new sites (in particular allosteric sites), which would otherwise be inaccessible, may become available for binding. The ability to capture the target protein in a specific conformation with high affinity for a significantly long time opens the possibility for a small-fragment molecule screening.

Biography:

E. Demet Akten is working on the protein dynamics of b2-adrenergic receptor, in order to understand the allosteric coupling behavior that exists between the intra- and extracellular parts of the receptor. She is also developing screening techniques for the same protein to better discriminate agonists from antagonists or inverse agonists. 

Abstract:

Background: This study investigates the allosteric coupling that exists between the intra- and extracellular parts
of human β2-AR, in the presence of intracellular loop 3 (ICL3), which is missing in all crystallographic experiments and most of the simulation studies. Our 1 μs long MD run has revealed a transition to an alternative inactive state of the receptor, in which ICL3 packed under G protein’s binding cavity and completely blocked its accessibility to G protein. Simultaneously, an outward tilt of transmembrane helix 5 (TM5) caused an expansion of the extracellular ligand-binding site. Independent runs with a total duration of 4 μs were carried out to further investigate this inactive state with packed ICL3 and the allosteric coupling event. Results: In all three independent unrestrained runs, ICL3 preserved its initially packed conformation during 500 ns long simulation, suggesting an inhibition of the receptor’s activity. Specific bond restraints were later imposed between some key residues at the ligand-binding site, which have been experimentally determined to interact with the ligand. Restraining the binding site region to an open state facilitated ICL3 closure, whereas a relatively constrained binding site hindered ICL3 packing. However, the reverse operation, i.e. opening of the packed ICL3, could not be realized by restraining the binding site region to a closed state. Thus, any attempt failed to free ICL3 from its locked state. Conclusions: Overall, our simulations indicated that starting with very inactive states, the receptor stayed almost irreversibly inhibited, which in turn decreased the overall mobility of the receptor. Bond restraints, which represented the geometric restrictions caused by ligands of various sizes when bound at the ligand-binding site, induced the expected conformational changes in TM5, TM6 and consequently, ICL3. Still, once ICL3 was packed, the allosteric coupling became ineffective due to strong hydrogen bonds connecting ICL3 to receptor’s core.

  • Track 8: Recent Advances in Structural Biology
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.

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 [1] 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 [2, 3]. 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 [3]. 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 [2].

In addition, we combine the new sample preparation method with a single cell lysis device for adherent eukaryotic cells [4] 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 [2]. 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 [5]. 

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.

Biography:

After her PhD at the University of Padua (Italy) Paola Picotti joined the group of Ruedi Aebersold at ETH Zuerich (Switzerland), where she developed novel targeted proteomic techniques. In 2011 she was appointed assistant professor at ETH Zurich. Her group develops structural and chemoproteomics methods and uses them to study the consequences of intracellular protein aggregation. Paola Picotti’s research was awarded an ERC Starting grant, a Professorship grant from the Swiss National Science Foundation, the Latsis Prize, the Robert J. Cotter Award, the SGMS Award and the EMBO Young Investigator Award. Main contributions of the Picotti group are the development of a structural method to analyze protein conformational changes on a system-wide level, the discovery of novel allosteric interactions, the analysis of the determinants of proteome thermostability and the identification of a novel neuronal clearance mechanism for a protein involved in Parkinson’s disease.

Abstract:

Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. Mass spectrometry (MS)-based proteomic techniques are routinely used to measure changes in protein abundance, post-translational modification and protein interactors, but much less is known about protein structural changes, owing to the lack of suitable approaches to study global changes in protein folds in cells.

 

In my talk I will present a novel structural proteomics technology developed by our group that enables the analysis of protein structural changes on a proteome-wide scale and directly in complex biological extracts. The approach relies on the coupling of limited proteolysis (LiP) tools and an advanced MS workflow. LiP-MS can detect subtle alterations in secondary structure content, larger scale movements such as domain motions, and more pronounced transitions such as the switch between folded and unfolded states or multimerization events. The method can also be used to pinpoint protein regions undergoing a structural transition with peptide-level resolution. I will describe selected applications of the approach, including 1. The identification of proteins that undergo structural rearrangements in cells due to a nutrient shift; 2. The analysis of in vivo protein aggregation; 3. The cell-wide analysis of protein thermal unfolding; and 4. The identification of protein-small molecule interactions (e.g drug-target deconvolution).

 

 I will discuss the power and limitations of the method and possible new directions in structural biology enabled by this emerging approach to protein structure analysis.

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.

Hejun Liu

China Novartis Institutes for Biomedical Research, China

Title: Structure-based discovery on the specificities of histone chaperone Hif1
Biography:

Hejun Liu has his expertise in structural biology and is focusing on histone chaperones and chromatin structure regulation. He is now an investigator at China Novartis Institutes for Biomedical Research. He has studied SHNi-TPR histone chaperone for years and solved histone chaperone Hif1 structure 3 years ago, which is the first structure of SHNi-TPR family. As he has said that the structure helps a lot in mapping the histone binding pattern of Hif1. Then he reconstituted various Hif1-histone complexes and solved the structure of Hif1 in complex with H2A-H2B. The complex structure revealed a novel chaperone specificity of Hif1 that interacts to both H2A-H2B dimer and H3-H4 tetramer.

Abstract:


Eukaryotic genomes are packed hierarchically into chromatins with nucleosomes as their basic repeat units. Histone chaperones involved in this process are critical for guiding specific histone post-translational modifications, safeguarding stepwise nucleosome assembly and disassembly, and thus regulating chromatin structures to change gene activities. Histone chaperone Hif1 (Hat1-interacting factor 1) was first reported to be involved in nuclear histone acetylation complex HAT-B that acetylates histone H4 at lysine K5 and K12 sites. In addition, Hif1 was also reported to be involved in telomere silence and nucleosome (dis)assembly. However, the structural basis of Hif1 and how it interacts to histones remains largely unknown. Hence we take our advantage in protein crystallographic research on Hif1 structure and its complex with core histones to reveal its chaperone specificities on core histones. We first solved the structure of Hif1 and revealed the architecture of SHNi-TPR (Sim3-Hif1-NASP interrupted tetratricopeptide repeat) proteins for the first time. The structure reveals that Hif1 contains a TPR domain formed by 4 TPR helix bundles and an interrupted coil-coiled acid loop that covers the rear surface of the TPR domain. Based on this structure, we demonstrated that both the TPR domain and acid loop are responsible for the histone binding. Hence, we solved the complex structure of Hif1-H2A-H2B-Hif1 to elucidate the chaperone specificity of Hif1. Based on these structure-based discoveries, our data revealed that Hif1 binds both H2A-H2B dimer and H3-H4 tetramer in an alternative binding manner. And this binding pattern is conserved across SHNi-TPR family from yeast to human. Overall, these findings provide clues for investigating the potential roles of SHNi-TPR proteins in nucleosome (dis)assembly.

Biography:

Karim Fahmy has his expertise in the field of spectroscopic investigations of membrane proteins. As an infrared spectroscopist, he has contributed to the understanding of fundamental molecular mechanisms of GPCR activation, using the visual photoreceptor, bovine rhodopsin, as a model system. His interest focuses on the functional role of the water/lipid/protein interface in GPCRs and other membrane proteins. Presently, he addresses the properties of water within membrane proteins and the linkage of membrane hydration to the protonation state of membrane protein side chains at the membrane interface. 

Abstract:

Statement of the Problem: The active transport of ions across biological membranes requires their hydration shell to interact with the interior of membrane proteins. Although restricted to the membrane protein surface, lipid protein interactions markedly affect transport rates of metals through P-type ATPases. However, the influence of the external lipid phase on internal dielectric dynamics is hard to access by experiment. Methodology & Theoretical Orientation:  Using the octahelical transmembrane architecture of the copper-transporting P1B-type ATPase from Legionella pneumophila as a model structure, we have established the site-specific labeling of internal cysteines with 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN), a polarity-sensitive fluorophore. This enabled dipolar relaxation studies in a solubilized form of the membrane protein and in its lipid-embedded state in nanodiscs. The latter provide a native-like lipid environment but facilitate spectroscopic studies due to their lower scattering in the UV-vis as compared to vesicles.  Conclusion & Significance: Time-dependent fluorescence shifts of BADAN linked specifically to either of the conserved copper-binding active site cysteines, Cys382 and Cys384 revealed the local hydration and dipole mobility around the transmembrane ion-binding motif. At Cys382, both parameters were strongly reduced upon lipid insertion of the membrane protein. The environment of Cys384, although less than a helical turn apart, was little affected by the lipidic phase. Against expectation and differently from Cys382, dipole mobility increased around Cys384 in NDs as compared to the less constrained detergent-solubilized state. Thus, the distribution of hydration and dipole mobility is shaped significantly and independently of each other by membrane lateral pressure. Vice versa, the lipidic phase may exert restoring forces on water-mediated H-bond networks around the conserved transmembrane ion-binding site for driving conformational changes during the catalytic transport cycle. 

Biography:

The Campbell lab at the University of North Carolina studies Ras superfamily GTPases as well as cell adhesion proteins that regulate cellular growth and motility. Our research employs a combination of biochemical, biophysical and structural biology approaches to characterize protein-protein and protein-ligand interactions, elucidate structures and characterize novel mechanisms of regulation.  Our structural and mechanistic studies of tumor promoter and tumor suppressor proteins, have led to the identification of novel effector interactions and post-translational modifications that drive Ras-mediated tumorigenesis, as well delineation of interactions critical for paladin, paxillin, focal adhesion kinase and vinculin-mediated cell motility. 

Abstract:

Vinculin (Vcn) is an essential cytoskeletal protein that acts as a scaffold to link transmembrane receptors to actin filaments, thereby playing a crucial role in cell adhesion, motility, and force transmission between cells. While Vcn is ubiquitously expressed, metavinculin (MVcn), a larger isoform of Vcn, is selectively expressed in smooth and cardiac muscle cells. Similar to Vcn, MVcn can directly associate with actin and remodel the actin cytoskeleton. However, distinct from Vcn, MVcn contains an additional exon that encodes a 68-residue insert.  Point mutations in the 68-residue insert have been associated with altered actin organization and heart disease. MVcn expression is higher in muscle cells that require greater force transmission. Given these observations, we postulate that MVcn plays an important role in force generation and transmission through its interaction with the actin cytoskeleton. The tail domains of Vcn (Vt) and MVcn (MVt) directly bind filamentous (F) actin and we have recently obtained ~8 Angstrom Cryo-EM reconstructions of Vt and MVt in complex with F-actin.  While Vt and MVt associate with F-actin through similar binding interfaces, they differ in their ability to reorganize actin filaments. We find that wild-type (WT) MVcn tail domain (MVt) does not bundle actin filaments, but the cardiomyopathy-associated MVt mutants show differences in actin filament bundling in vitro. We are currently investigating how MVcn regulates actin organization in the presence of Vcn. While we have observed that WT MVt inhibits Vt-mediated actin bundling, the disease-associated MVt mutants fail to inhibit Vt-mediated actin bundling via negative-stain EM. We have additionally succeeded in expressing MVcn in Vcn null mouse embryonic fibroblasts (MEF) and observed that MVcn localizes to focal adhesions, similar to Vcn. Results from these studies will provide the groundwork for how MVcn disease mutants contribute to associated cardiomyopathies.