Day 3 :
- Track 10: Structural Biology in Cancer Research | Track 11; Current Trends
Gordon Life Science Institute, Boston, USA
Guo-Ping Zhou is currently a Distinguished Professor of Gordon Life Science Institute, USA. He is also an Adjunct Professor of several academics in the United States and China. Dr. Zhou received his Ph.D in Biophysics from University of California at Davis, and completed his postdoctoral training at Stanford University and Harvard University, respectively. Dr. Zhou determined the 3D NMR structures of some important proteins, protein-DNA complexes, and super lipids. He has successfully introduced the elegant wenxiang diagrams to elucidate the biological mechanisms of protein-protein/ligands interactions observed by NMR. Meanwhile, he has also published many papers in bioinformatics, and edited some special issues on structural biology for several influential scientific journals.
The α2,8-sialyltransferase (ST8Sia) family consists of 6 sia-lytranseferases, which are related to forms of polysialic acid chains (PSA) on neural cell adhesion molecule (NCAM) and NCAM polysialylation, and have important effects on formation of sialic acid storage diseases, neural system diseases and invasive cancers. It has been known that synthesis of PSA chains is catalysed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST). In addition, a polybasic motif of 32 amino acids in both ST8Sia II and ST8Sia IV has been designated as “polysialytransferase domain” (PSTD), which is essential for NCAM polysialylation. In this study, we have determined the 3D structure of the PSTD peptide containing 22 amino acids (22AA) in ST8Sia IV using NMR spectroscopy. This NMR-based model displays that the PSTD domain consists of an α-helical segment, two unstructured domains in both N- and C-terminus, and two three-residue-loops near the C-terminus of the peptide. Our overlaid 2D 1H-15N-HSQC spectra of the 22AA-PSTD peptide show that the amide proton chemical shifts of some amino acids such as I260, I261, H262, R265, L269 and K272 have been changed after polySA was mixed with the PSTD peptide. In addition, the peak intensity of A263, V264, R265, Y267, L269 and K272 were also decreased after adding polySA. However, there is no any change in both chemical shift and the amide proton peak intensity for all other residues located on outside of the helix. Above NMR results indicate a weak interaction exists between the helix of the PSTD and the PolySA, which may play a vital roles in modulating biosynthesis of polySA chain and NCAM polysialylation.
- Huang RB, Cheng D, Lu B, Liao SM Troy li, FA, Zhou GP (2017) The Intrinsic Relationship between Structure and Function of the Sialytransferase ST8Sia Family Members. Curr. Top. Med. Chem. 7(21):2359-2369.
- Zhou G P, W Z Zhong (2016). Perspectives in the medicinal chemistry. Current Topics in Medicinal Chemistry.16(4):381-382.
- Zhou G P (2016) Modulations and their biological functions of protein-biomolecule interactions. Current Topics in Medicinal Chemistry. 16(6):579-580.
- Zhou G P, Chen D, Liao S, Huang R B (2016) Recent progresses in studying helix-helix interactions in proteins by incorporating the Wenxiang diagram into the NMR spectroscopy. Current Topics in Medicinal Chemistry, 16(6)581-590.
- Zhou G P, Huang R B, Troy, F A (2015) 3D Structural Con formation and Functional Domains of Polysialyltransferase ST8Sia IV Required for Polysialylation of Neural Cell Adhesion Molecules. Protein Peptide Lett. 22(2):137-148.
Istanbul Medipol University, Turkey
As being a computational biophysicist, my research has focused on understanding underlying molecular mechanisms of biologically important problems and also providing mechanistic insight at the molecular level. In particular, I have been working with GPCRs and their interacting partners which are responsible for cellular signaling. In order to complement relevant experimental studies one needs to access long time-scales and big system sizes which are beyond the classic MD simulations. In this respect, my expertise in doing long-time MD simulations and application of enhanced sampling techniques such as accelerated MD, metadynamics, and steered MD which provides a good fit. I work in close collaboration with medicinal chemists to direct them for effective molecular designs. In addition, I am also responsible for testing the efficacy of these molecules in silico before transferring them to either in vitro or in vivo studies. Recently, I have been awarded an international COST (European Cooperation in Science and Technology) grant which is based on developing heterobivalent molecules capable of binding more than one target for treatment of symptoms of Parkinson’s disease.
Arrestins (Arrs) are a family of four proteins (Arr1- 4) which mediate G-protein-coupled receptor (GPCR) desensitization and internalization by coupling to active and phosphorylated receptor. Recently, they have also been shown to mediate GPCR-independent signaling pathways. The specific functions of Arrs (desensitization vs. G-protein-independent signaling) can be regulated by differential phosphorylation of the receptor, which is known as the phosphorylation barcode. The molecular mechanism responsible for formation of a high-affinity complex between an Arr subtype and a GPCR having a certain phosphorylation pattern remains elusive but is crucial for directing the subtype towards a specific functional role, and hence paves the way for development of safer therapeutics with fewer side-effects. As a first step in that direction, we have started with elucidating the activation mechanism of Arr subtypes by carrying out comparative molecular dynamics (MD) studies of the two members of the family, namely Arr1 and Arr3, which exhibit the largest differences in terms of phosphorylation selectivity. In addition, we also modeled and simulated Arr1-R175E mutant, which is known to be constitutively active, and compared it to Arr1 and Arr3 to detect activation-related rearrangements. We found novel structural elements that had not been considered before as determinants for activation and can be targeted with drugs for functional modulation. The emerging model also proposes that activation of Arr1-R175E is connected to perturbation of the well-known region, namely, the polar-core, whereas no changes were observed in that region in Arr3 in spite of the presence of other activation-related changes. With that, we could propose a structural model to explain the molecular mechanism responsible for markedly reduced selectivity of Arr3 towards phosphorylated GPCRs. Finally, knowledge achieved in this study can also be utilized to modulate Arr binding to GPCRs under disease conditions such as otozomal dominant disorders and congestive heart failure.
- Sensoy O, Atılgan A R and Atılgan C (2017) FbpA iron storage and release are governed by periplasmic microenvironments. PCCP. 19(8): 6064-6075.
- Sensoy O, Moreira I S and Morra G (2016) Understanding the Differential Selectivity of Arrestins toward the Phosphorylation State of the Receptor. ACS Chemical Neuroscience. 7(9):1212.
- Arango Lievano M, Sensoy O, Borie A, Corbani M, Guillon G, Sokoloff P and Weinstein H, Jeanneteau F (2015) A GIPC1-Palmitate Switch Modulates Dopamine DRD3 Receptor Trafficking and Signaling. Mol. Cell. Biol. 36(6):1019-1031.
- Sensoy O and Weinstein H (2015) A mechanistic role of Helix 8 in GPCRs: Computational modeling of the Dopamine D2 Receptor interaction with the GIPC1-PDZ domain. BBA-Biomembranes. 1848(4):976-983.
- Dalgicdir C Sensoy O and Sayar M (2013) A transferable coarse-grained model for diphenylalanine: how to represent an environment driven conformational transition. J. Chem. Phys. 139(23):234115.
Merck Research Laboratories, USA
Stephen Soisson, PhD is a director of Biochemical Engineering and Structure at Merck Research Laboratories in West Point, Pennsylvania (USA). With 25+ years of structural biology experience, Dr. Soisson has focused research on elucidating the structural aspects of biological regulatory mechanisms, and applying these insights in the area of structure-based drug design. He has served on the scientific advisory boards of the Structural Genomics Consortium, and the GPCR Consortium.
Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Recently identified, full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. Our recent studies have led to a 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM. Surprisingly, the structure reveals a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5). These structural rearrangements, along with AgoPAM binding, appear to trigger the transition of intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.
- NL Elsen, SB Patel, RE Ford, DL Hall, F Hess, H Kandula, M Kornienko et. al. (2017) Insights into activity and inhibition from the crystal structure of human O-GlcNAcase. Nature Chemical Biology. 13 (6):613-615.
- H Zhang, GW Han, A Batyuk, A Ishchenko, KL White, N Patel et. al. (2017) Structural basis for selectivity and diversity in angiotensin II receptors. Nature. 544(7650):327-332.
- HP Su K Rickert, C Burlein, K Narayan, M Bukhtiyarova, DM Hurzy, et. al. (2017) Structural characterization of nonactive site, TrkA-selective kinase inhibitors. Proceedings of the National Academy of Sciences. 114(3):297-306.
- M Scheepstra, S Leysen, GC Van Almen, JR Miller, J Piesvaux, V Kutilek, et. al. (2015) Identification of an allosteric binding site for RORγt inhibition. Nature communications. 6:8833.
UMass Medical School, USA
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
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.
- Demo G, Svidritskiy E, Madireddy R, Diaz-Avalos R, Grant T, Grigorieff N, Sousa D, Korostelev AA. Mechanism of ribosome rescue by ArfA and RF2. preprint in bioRxiv. 2016 Dec. 2. (animation)
- Loveland AB, Bah E, Madireddy R, Zhang Y, Brilot AF, Grigorieff N, Korostelev AA. Ribosome•RelA structures reveal the mechanism of stringent response activation. eLife. 2016 July 19. (animation) animation)
- Abeyrathne PD, Koh CS, Grant T, Grigorieff N, Korostelev AA. Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome. eLife. 2016 May 9. (animation)
- Svidritskiy E, Madireddy R, Korostelev AA. Structural Basis for Translation Termination on a Pseudouridylated Stop Codon. J Mol Biol. 2016 Apr 20.
- Svidritskiy E, Korostelev AA. Ribosome Structure Reveals Preservation of Active Sites in the Presence of a P-Site Wobble Mismatch. Structure. 2015 Nov 3;23(11):2155-61.
Academia Sinica, Taiwan
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.
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.
- Pan KT, Chen YY, Pu TH, Chao YS, Yang CY, Bomgarden RD, Rogers JC, Meng, TC*, Khoo, KH* (2014) Mass spectrometry based quantitative proteomics for dissecting multiplexed redox cysteine modifications in nitric oxide-protected cardiomyocyte under hypoxia. Antioxidant and Redox Signaling, 20:1365-1381.
- Santhanam A, Peng WH, Yu YT, Sang TK, Chen GC*, Meng TC* (2014) Ecdysone-induced receptor tyrosine phosphatase PTP52F regulates Drosophila midgut histolysis by enhancement of autophagy and apoptosis. Mol. Cell. Biol., 34:1594-1606.
- Chen KE, Lin SY, Wu M J, Ho MR, Santhanam A, Chou CC, Meng TC*, Wang AHJ* (2014) Reciprocal allosteric regulation of p38γ and PTPN3 involves a PDZ domain modulated complex formation. Science Signaling, 7: ra98 p1-12.
- Chen KE, Li MY, Chou CC, Ho MR, Chen GC, Meng TC*, Wang AHJ* (2015) Substrate specificity and plasticity of FERM-containing protein tyrosine phosphatases. Structure, 23: 653–664.
- Hsu MF, Pan KT, Chang FY, Khoo KH, Urlaub H, Chang GD*, Haj FG*, and Meng TC* (2016) S-Nitrosylation of endogenous protein tyrosine phosphatases in endothelial insulin signaling. Free Rad Biol Med, 99: 199-213.
Max Planck Institute of Biochemistry, Germany
Christian Biertümpfel obtained his Ph.D. degree from the European Molecular Biology Laboratory (EMBL) and the Ruprecht Karls University of Heidelberg, Germany. His Ph.D. research focused on the crystallization and characterization of Holliday junction resolvases. During his postdoctoral time at the National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, USA, he was able to solve a first crystal structure of a Holliday junction resolvase from T4 phages in complex with a DNA four-way junction. Furthermore, together with Wei Yang he determined the structure and mechanism of human DNA polymerase η functioning as a molecular splint. After a short period at the Vaccine Research Center, NIAID, NIH, he moved to the Max Planck Institute of Biochemistry, Martinsried, Germany as a Max Planck Research Group Leader. Recently, the Biertümpfel lab obtained structural information on the human Holliday junction resolvase GEN1 and they found for the first time a chromodomain extending a nuclease domain.
Several DNA repair and maintenance pathways depend on the correct and efficient processing of DNA intermediates by structure-specific nucleases. Human Holliday junction resolvase GEN1 seems to be an enzyme of last resort for recognizing and cleaving a specific range of DNA structures. The crystal structure of human GEN1 in complex with Holliday junction DNA pinpointed to a crucial role of the chromodomain for efficient DNA recognition and cleavage. We further characterized different DNA-binding modes of GEN1 using biochemical methods in combination with structure-guided mutagenesis. The analysis highlights the importance of the arch region to distinguish between different DNA substrates. In addition, we identified a cluster of positive amino acids shadowing the chromodomain to assist the enzyme for robust DNA recognition. Moreover, we directly show that GEN1 operates as a monomer with 5’ flap DNA and as a dimer in complex with DNA four-way junctions, which is a unique feature in the Rad2/XPG nuclease family. This linked cleavage mechanism ensures that DNA junctions are resolved in a strictly symmetric manner without altering DNA information. GEN1’s DNA recognition features make it a versatile tool for DNA processing and for maintaining genome integrity.
Figure: Holliday junction resolvase GEN1 is a monomer in solution and thus, cleavage competent for 5’ flap substrates. However, it can only cleave DNA four-way junctions by forming an active nuclease dimer.
- Lee SH, Princz LN, Klügel MF, Habermann B, Pfander B, Biertümpfel C. (2015). Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage. Elife, 4, e12256.
- Zhao Y, Gregory MT, Biertümpfel C, Hua YJ, Hanaoka F, Yang W. (2013). Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η. Proc Natl Acad Sci U S A 110, 8146-51.
- Joyce MG, Kanekiyo M, Xu L, Biertümpfel C, Boyington JC, Moquin S, Shi W, Wu X, Yang Y, Yang ZY, Zhang B, Zheng A, Zhou T, Zhu J, Mascola JR, Kwong PD, Nabel GJ. (2013). Outer domain of HIV-1 gp120: antigenic optimization, structural malleability, and crystal structure with antibody VRC-PG04. J Virol 87, 2294-306.
- Zhao Y, Biertümpfel C, Gregory MT, Hua YJ, Hanaoka F, Yang W. (2012). Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin. Proc Natl Acad Sci U S A 109, 7269-74.
- Hansman GS, Biertümpfel C, Georgiev I, McLellan JS, Chen L, Zhou T, Katayama K, Kwong PD. (2011). Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability. J Virol 85, 6687-701
ETH Zurich, Switzerland
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.
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.
- Leuenberger P, Ganscha S, Kahraman A, Cappelletti V, Boersema P,J, von Mering C, Claassen M, Picotti P. Cell-wide analysis of protein thermal stability across species reveals the determinants of thermostability, Science, (in press).
- Feng Y, De Franceschi G, Kahraman A, Soste M, Melnik A, Boersema PJ, de Laureto PP, Nikolaev Y, Oliveira AP, Picotti P. Global analysis of protein structural changes in complex proteomes. Nat Biotechnol. 2014; 32(10):1036-44.
- Soste M, Hrabakova R, Wanka S, Melnik A, Boersema P, Maiolica A, Wernas T, Tognetti M, von Mering C, Picotti P. A sentinel protein assay for simultaneously quantifying cellular processes. Nat Methods. 2014; 11(10):1045-8.
- Picotti P, Clément-Ziza M, Lam H, Campbell DS, Schmidt A, Deutsch EW, Röst H, Sun Z, Rinner O, Reiter L, Shen Q, Michaelson JJ, Frei A, Alberti S, Kusebauch U, Wollscheid B, Moritz RL, Beyer A, Aebersold R. A complete mass-spectrometric map of the yeast proteome applied to quantitative trait analysis. Nature. 2013; 14;494(7436):266-70.
- Picotti P, Bodenmiller B, Mueller LN, Domon B, Aebersold R. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell. 2009; 21;138(4):795-806.
Weizmann Institute of Science, Israel
Hagen Hofmann received his PhD from the Martin-Luther University Halle-Wittenberg (Germany) in 2008. In the period 2008 - 2014, he was a postdoctoral fellow at the University of Zurich in the group of Benjamin Schuler and since 2014 he is heading the “Molecular Systems Biophysics” group at the Weizmann Institute of Science (Israel). He and his group use a broad set of single-molecule fluorescence tools to understand the dynamics of proteins and protein networks on timescales from nanoseconds to hours. In addition, live-cell imaging, in vivo single-molecule FRET, and single particle tracking is used to monitor proteins in live cells. His interest ranges from the physics of disordered proteins over coupled binding and folding reactions up to stochastic genetic circuits and regulatory protein networks.
Conformational transitions in proteins are typically captured well by rate equations that predict exponential kinetics for two-state reactions. Here, we describe a remarkable exception. The electron-transfer enzyme quiescin sulfhydryl oxidase (QSOX), a natural fusion of two functionally distinct domains, switches between open and closed domain arrangements with apparent power law kinetics. Using single-molecule Foerster resonance energy transfer (FRET) experiments on timescales from nanoseconds to milliseconds, we show that the unusual open-close kinetics results from slow domain rearrangements in a heterogeneous ensemble of open conformers. While substrate accelerates the kinetics, thus suggesting a substrate-induced switch to an alternative free energy landscape of the enzyme, the power-law behavior is also preserved upon electron load. Our results show that conformational multiplicity with slow sampling dominates the motions of QSOX, thus providing an explanation for catalytic memory effects in other enzymes.
- Grossman, I. et al. (2015) Single-molecule spectroscopy exposes hidden states in an enzymatic electron relay. Nature Communications 6:1-10.
- Hofmann, H (2016). Speedy Motion For Function. Nature Chemical Biology. 12:576-577.
- Schuler, B; Soranno, A; Hofmann, H; Nettels, D (2016). Single-Molecule Fret Spectroscopy and the Polymer Physics of Unfolded and Intrinsically Disordered Proteins. Annual Review of Biophysics. 45:207-231.
- Hofmann, H; Soranno, A; Borgia, A; Gast, K; Nettels, D; Schuler, B (2012). Polymer Scaling Laws of Unfolded and Intrinsically Disordered Proteins Quantified With Single-Molecule Spectroscopy. Proceedings of the National Academy of Sciences of the United States of America. 109:16155-16160.
5. Hofmann, H; Hillger, F; Pfeil, Sh; Hoffmann, A; Streich, D; Haenni, D; Nettels, D; Lipman, Ea; Schuler, B (2010). Single-Molecule Spectroscopy of Protein Folding in a Chaperonin Cage. Proceedings of the National Academy of Sciences of the United States of America. 107:11793-11798.
University of Angers, France
Marie Chabbert is a scientist from the French CNRS (Centre National de la Recherche Scientifique). She has her expertise in molecular modeling and bioinformatics approaches to the structure-function relationship of proteins. She has special interest in deciphering the mechanisms that drove protein evolution and in using evolutionary data to gain structural and functional information on protein families. She is presently working on the G protein-coupled receptors, especially chemotaxic and vasoactive peptide receptors.
Statement of the Problem: Co-variations between positions in a multiple sequence alignment may reflect structural, functional, and/or phylogenetic constraints. Numerous co-variation methods have been developed and may yield a wide variety of results. However, few studies have been undertaken to determine co-variations methods adequate to gain information on functional divergence within a protein family. Methodology & Theoretical Orientation: We explore co- variation methods for their capability to mine co-varying positions related to the functional divergence in a protein family. To reach this objective, we compare several methods on a model system that consists of three nested sets of about 300, 100, and 20 paralogous sequences of a protein family, the class A of G protein-coupled receptors. The co- variation methods analyzed are based on chi2 scores, mutual information, substitution matrices, or perturbation methods. We analyze the dependence of the co-variation scores on residue conservation, measured by sequence entropy, and the networking structure of the top pairs. Findings: Out of the four methods that privilege top pairs with intermediate entropy, two favor individual pairs, whereas the other two methods, OMES (Observed minus Expected Squared) and ELSC (Explicit Likelihood of Subset Covariation), favor a network structure with a central residue involved in several high scoring pairs. This network structure is observed for the three sequence sets, making a hierarchical analysis possible. In each case, the central residue corresponds to a residue known to be crucial for the evolution of the protein family and the sub-family specificity. Positions co-varying with this central residue form a few clusters in the receptor 3D structure (Fig. 1). Conclusion & Significance: The central residues obtained with the OMES or ELSC methods can be viewed as evolutionary hubs, in relation with an epistasis-based mechanism of functional divergence within a protein family.
- Chantreau V., Taddese B., Munier M., Gourdin L., Henrion D., Rodien P. and Chabbert M. (2015) Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor, Plos One, 10(11):e0142250.
- Pelé J., Moreau M., Abdi H., Rodien P., Castel H. and Chabbert M. (2014) Comparative analysis of sequence co-variation methods to mine evolutionary hubs: Examples from selected GPCR families. PROTEINS 82:2141-56.2013.
- Pelé J., Bécu J.-M., Abdi H. and Chabbert M. (2012) Bios2mds: an R package for comparing orthologous protein families by metric multidimensional scalingBMC Bioinformatics 13, 133.
- Chabbert M., Castel H., Pelé J., Devillé J., Legendre R. and Rodien P. (2012) Evolution of Class A G-Protein-Coupled Receptors: Implications for Molecular Modeling. Curr. Med. Chem. 19, 1110-8.
- Pelé J., Abdi H., Moreau M., Thybert D., and Chabbert M. (2011) Multidimensional scaling reveals the main evolutionary pathways of class A G-protein-coupled receptors. (2011) PLoS ONE 6, e19094.
Agency for Science, Technology and Research (A*STAR), Singapore
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.
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.
- Zhang Z, Li Y, Loh YR, Phoo WW, Hung AW, Kang C*, Luo D* (2016) Crystal structure of unlinked NS2B-NS3 protease from Zika virus. Science, 354(6319):1597-1600.
- W. W. Phoo, Y. Li, Z. Z. Zhang, M. Y. Lee, Y. Loh, Y. B. Tan, E. Y. Ng, J. Lescar, C. Kang*, D. Luo* (2016) Structure of NS2B-NS3 Protease from Zika Virus after Self-cleavage. Nature communications, 7:13410.
- Y. Li, YL Wong, M.Y. Lee, Q. Li, J. Lescar, P.Y. Shi, C. Kang*, (2016) Secondary structure and membrane topology of the full length Dengue NS4B in micelles. Angewandte Chemie Int Ed Engl, 55(39):12068-72
- Y. Li, Q. Li, Y.L. Wong, L. Liew, and C.B. Kang,*(2015) Membrane topology of NS2B of dengue virus revealed by NMR spectroscopy. BBA-Biomembranes. 1848: 2244-2252.
- Zou J, Xie X., Lee LT., Chandrasekaran R., Reynaud A., Yap L., Wang Q., Dong H., Kang C.B., Yuan Z., Lescar J., and Shi P. (2014) Dimerization of Flavivirus NS4B Protein. Journal of Virology. 88(6): 3379-3391.
Johns Hopkins University, USA
The research focus in the Sohn is to understand the molecular mechanism by which human immune system engages invading pathogens. Jay received B.S. from University of Michigan, and Ph.D. from Duke University. Upon completing his post-doctoral training at MIT with Dr. Bob Sauer, he joined the faculty at Johns Hopkins in 2011.
Absent-in-melanoma-2-like receptors (ALRs) detect foreign double-stranded (ds)DNA from invading pathogens and assemble into filamentous signaling platforms termed inflammasome. The ALR filaments play crucial roles in launching antiviral and inflammatory responses against a number of pathogens (e.g. HIV and HSV); however, persistent ALR complexes are also linked to autoimmune disorders (e.g. Sjögren’s syndrome and lupus). Here, by combining solution assays, electron microscopy, and single-molecule methods, we investigate the filament assembly mechanisms of two prototypical ALRs, namely IFI16 and AIM2.
(1) IFI16 detects foreign dsDNA both in the host nucleus and cytoplasm. We found that IFI16 uses dsDNA as a one-dimensional diffusion-scaffold to assemble into filaments. The dsDNA-binding HIN200 domains of IFI16 are responsible for tracking dsDNA, while its pyrin domain (PYD) is necessary for filament assembly. Importantly, nucleosomes represent barriers that prevent IFI16 from targeting host dsDNA by directly interfering with its assembly. This unique scanning-assisted assembly mechanism would allow IFI16 to distinguish self- from nonself-dsDNA in the nucleus.
(2) AIM2 detects cytoplasmic dsDNA and assembles into an inflammasome. We found that the PYD of AIM2 (AIM2PYD) drives both filament formation and dsDNA binding. As with IFI16, the size of exposed dsDNA acts a key regulator for the polymerization of AIM2. The helical symmetry of the upstream AIM2PYD filament is consistent with the filament assembled by the PYD of the downstream ASC adaptor, indicating that AIM2 acts as a structural template for polymerizing ASC.
Together, our studies provide a unifying paradigm for how ALRs carry out foreign dsDNA-sensing pathways, where generating a structural template by coupling ligand-binding and oligomerization plays a key signal transduction mechanism.
- Morrone, S.M., Wang, T., Constantoulakis, L.M., Hooy, R.M., Delannoy, M.R., and Sohn, J. (2014) Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy. PNAS 111, E62-71
- Geertsma, H.J., Schute, A.C., Spenkelink, L.M., Mcgrath, W.J., Morrone, S.R., Sohn, J., Mangel, W.F., Robinson, A., van Oijen, (2015) A. Single-molecule imaging at high fluorophore concentrations of Dye (LaDYe). Biophysical Journal 108, 949-56.
- Baer, A.N., Petri, M.A., Sohn, J., Rosen, A., Casciola-Rosen, L. Antibodies to human IFI16 are present in systemic lupus and primary Sjögren’s syndrome with similar frequencies but detect different parts of molecule. (2015) Arthritis Care Res.
- Morrone, S.M. Matyszewski, M., Yu, X., Delannoy, M., Egelman, E.H., and Sohn J. Assembly driven activation of the AIM2 inflammasome provides a template for the polymerization of downstream ASC. (2015) Nature Communications. 6, 7827.
- Stratmann, S., Morrone, S.M., van Oijen, A.M.*, and Sohn, J*. The innate immune sensor IFI16 recognizes foreign DNA in the nucleus by scanning along the duplex. (2015). eLife e11721 *: co-corresponding authors
University of Massachusetts Medical School, USA
Despite the importance of RNA-binding proteins to gene regulation, our understanding of how their structure and dynamics contribute to their biological activity is limited. In this study, we focus on two related RNA-binding proteins—TTP and TIS11d—that regulate the stability of mRNA transcripts encoding key cancer-related proteins, such as tumor necrosis factor-a and vascular endothelial growth factor. These two proteins display differential folding propensity in the absence of RNA, despite sharing a high sequence identity. We identified three residues located at the C-terminal end of an a-helix that determine the folding propensity of the RNA-binding domain in the apo state. We also showed that stabilization of the structure of the RNA-binding domain is associated with differences in RNA-binding activity in vitro and increased RNA-destabilizing activity in the cell. Phylogenetic analysis indicates that this family of proteins has only recently evolved to be able to modulate its biological activity through its dynamic structure.
To investigate how three residues determine the folding and stability of the TZF domain we used molecular dynamics and NMR spectroscopy. We observed that a p-p stacking between the side chains of a conserved phenylalanine and the zinc coordinating histidine is essential to maintain the correct tetrahedral geometry between the three cysteines, the histidine and the zinc ion. A hydrogen bond in the C-terminal zinc finger of TIS11d is important to keep the phenylalanine in proximity of the imidazole ring of the zinc coordinating histidine in a conformation that allows for stacking of the side chains. Lack of this hydrogen bond in TTP is responsible for the reduced zinc affinity of the C-terminal zinc finger. Sequence alignment shows that this phenylalanine residue is highly conserved. These results suggest that most CCCH-type zinc finger proteins employ p-p interactions to stabilize the structure of the TZF domain.
Figure 1: The stacking of the aromatic rings of the conserved Phe and of the zinc coordinating His stabilizes conformation of the His in a rotameric state compatible for zinc-binding.
- Morgan, B. R.; Massi, F. (2010) A computational study of RNA binding specificity in the tandem zinc finger domain of TIS11d. Protein Sci. 19: 1222-1234
- Morgan, B. R.; Massi, F. (2010) Accurate estimates of free energy changes in charge mutations. J. Chem. Theory Comput. 6: 1884-1893.
- Morgan, B. R.; Deveau, L. M.; Massi, F. (2015) Probing the structural and dynamical effects of the charged residues of the TZF domain of TIS11d. Biophys. J. 108: 1503-1515.
- Tavella, D.; Deveau, L. M.; Massi, F. (2016) Understanding the origin of the disorder of the tandem zinc finger domain of TTP. J. Chem. Theory Comput. 12: 4717-4725.
- Deveau, L. M.; Massi, F. (2016 ) Three residues make an evolutionary switch for folding and RNA-destabilizing activity in the TTP family of protein. ACS Chem. Biol. 11: 435-43.
Federal University of Rio de Janeiro, Brazil
Title: Role of conformational equilibrium in molecular recognition and capsid assembly: the case of flavivirus capsid proteins
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).
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.
- Iqbal, A., Moraes, A. H., Valente, A. P. & Almeida, F. C. L. Structures of the reduced and oxidized state of the mutant D24A of yeast thioredoxin 1: insights into the mechanism for the closing of the water cavity. J. Biomol. NMR 63, 417–423 (2015).
- de Paula, V. S., Razzera, G., Barreto-Bergter, E., Almeida, F. C. L. & Valente, A. P. Portrayal of complex dynamic properties of sugarcane defensin 5 by NMR: multiple motions associated with membrane interaction. Structure 19, 26–36 (2011).
- Samsa, M. M. et al. Dengue virus capsid protein usurps lipid droplets for viral particle formation. PLoS Pathog. 5, e1000632 (2009).
- Faustino, A. F. et al. Dengue virus capsid protein interacts specifically with very low-density lipoproteins. Nanomedicine 10, 247–55 (2014).
- Martins, I. C. et al. The disordered N-terminal region of dengue virus capsid protein contains a lipid droplet-binding motif. Biochem. J. 444, 405–415 (2012).
Yale University, USA
Sushant Kumar is a post-doctoral associate in the molecular biophysics and biochemistry department at the Yale university. He has extensive experience in biological data mining, proteins simulations and cancer genomics. He is particularly interested in integrating genomic variation data and protein structural data to develop novel methods assessing disease variant impact. In past, he has applied coarse-grained models to decipher the role of various physical factors influencing the coupled folding and binding mechanism observed among disordered proteins.
Statement of the Problem: The exponential rise in next-generation sequencing data is presenting considerable challenges in terms of variant interpretation. Though deep sequencing is unearthing large numbers of rare single nucleotide variants (SNVs), the rarity of these variants makes it difficult to evaluate their potential deleteriousness with conventional phenotype-genotype associations. Furthermore, many disease-associated SNVs act through mechanisms that remain poorly understood. 3D protein structures may provide valuable substrates for addressing these challenges. We present two general frameworks for doing so. In our first approach, we use localized frustration, which quantifies unfavorable residue interactions, as a metric to investigate the local effects of SNVs. In contrast to this metric, previous efforts have quantified the global impacts of SNVs on protein stability, despite the fact that local effects may impact functionality without disrupting global stability (e.g. in relation to catalysis or allostery). In our second approach, we employ models of conformational change to identify key allosteric residues by predicting essential surface pockets and information-flow bottlenecks (a new software tool that enables this analysis is also described). Importantly, although these two frameworks are fundamentally structural in nature, they are computationally efficient, thereby making analyses on large datasets accessible. We detail how these database-scale analyses shed light on signatures of conservation, as well as known disease-associated variants, including those involved in cancer.
Figure 2: The effect of introducing a typical deleterious SNV (∆F < 0). Each of the two vertical lines represents an energy-level diagram. Each level on this energy scale corresponds to the total energetic value of the protein if the residue position) were to be occupied by distinct amino acids. The ∆F associated with an SNV is negative if the SNV introduces a destabilizing effect.
- Kumar S, Clarke D, Gerstein M(2016) Localized structural frustration for evaluating the impact of sequence variants. Nucleic Acid Research 429:435-445.
- Clarke D, Sethi A, Li Shantao, Kumar S, Chang RW, Chen J, Gerstein M (2016) Identifying Allosteric Hotspots with Dynamics: Application to Inter- and Intra-species Conservation. Structure 24:826-37.
- Sethi A, Clarke D, Chen J, Kumar S, Galeev TR, Regan L, Gerstein M (2015) Reads meet rotamers: structural biology in the age of deep sequencing. Current Opinion in Structural Biology 35:125-34.
University of Sheffield, UK
Abdulrahman Alshehri working at security Forces Hospital, Riyadh, Saudi Arabia for 12 years. I did my Phd at sheffield University, UK. I have been working in creating long acting therapies using different stratigies such as, glycosylated linkers. Using multiple techniquies like PCR, gene cloning, cell culture, pharmacodynamic and pharmacokinetic to generate these therapies.
Rationale: The current therapeutic drugs such as, growth hormone (GH), granulocyte colony-stimulating factor (GCSF) and leptin require once-daily injections, which are inconvenient and expensive. Therefore, a number of approaches to reducing therapeutic regimens clearance have been tried mainly through conjugation with another moiety. One such technology already being employed is PEGylation; however this has been shown to be non-biodegradable and toxic. A previous study by Asterion has shown that the use of glycosylated-linkers between two GH ligands to create protein-tandems resulted in their glycosylation and an increased molecular weight (MW) whilst maintaining biological activity. The use of this technology using GCSF as an example will be presented, but can be easily applied to other molecules such as leptin.
Hypothesis: The incorporation of variable glycosylated linkers between two GCSF ligands will create a construct with high molecular weight and protected from proteolysis resulting in reduced clearance with out blocking bioactivity.
Methodology: GCSF tandems with linkers containing between 2-8 NAT glycosylation motifs and their respective controls (Q replaces N in the sequence motif NAT so there is no glycosylation) were cloned, and sequenced. Following expression in Chinese hamster ovary (CHO) cells, expressed protein was analysed by SDS-PAGE to confirm molecular weights. In vitro bioactivity was tested using an AML-193 proliferation assay. Immobilised Metal Affinity Chromatography (IMAC) was used to purify the protein. Pharmacokinetic and pharmacodynamics properties of the purified GCSF tandem proteins were measured in normal Sprague Dawley rats with full ethical approval.
Results: Purified glycosylated tandems show increased molecular weight above that of controls when analysed by SDS-PAGE. All GCSF tandems show increased bioactivity in comparison to native GCSF. Following intravenous administration to rats, GCSF2NAT, GCSF4NAT, GCSF8NAT containing 2, 4 & 8 glycosylation sites respectively and GCSF8QAT (non‐glycosylated GCSF tandem control) showed approximately 3‐fold longer circulating half‐life compared to that reported for the native GCSF (1.79 hours). Both GCSF2NAT and GCSF4NAT show a significant increase in the percentage of neutrophils over controls at 12 hours post injection. This effect however is short lived as the counts at 24+ hours are not significantly different to controls. GCSF8NAT shows an increase in the percentage of neutrophils that is only significant at 48 hours.
Conclusion: Results show that the use of glycosylated linkers to generate GCSF tandems results in molecules with increased molecular weight, improved in vitro bioactivity, longer circulating half-lives and enhanced neutrophilic population when compared to both native GCSF and the non-glycosylated tandem protein.
Figure 1: An example of 2NAT glycosylation motifs and its control 2QAT within a flexible linker (Gly4Ser)n between two GCSF ligands. (A) The glycosylation motif 2NAT inserted to the linker (glycosylated linker). (B) Non-glycosylation motif 2QAT control.
- LI & D'ANJOU 2009. Pharmacological significance of glycosylation in therapeutic proteins. Curr Opin Biotechnol, 20, 678-84.
- SINCLAIR & ELLIOTT 2005. Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. J Pharm Sci, 94, 1626-35.
- TANAKA et al. 1991. Pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to polyethylene glycol in rats. Cancer Res, 51, 3710-4.
- CHUNG, H. K., KIM, S. W., BYUN, S. J., KO, E. M., CHUNG, H. J., WOO, J. S., YOO, J. G., LEE, H. C., YANG, B. C., KWON, M., PARK, S. B., PARK, J. K. & KIM, K. W. 2011. Enhanced biological effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant, on HL60 cells. BMB Rep, 44, 686-91.
- COOPER, K. L., MADAN, J., WHYTE, S., STEVENSON, M. D. & AKEHURST, R. L. 2011. Granulocyte colony-stimulating factors for febrile neutropenia prophylaxis following chemotherapy: systematic review and meta-analysis. BMC Cancer, 11, 404.
- Young Researchers Forum
Jessica Thomaston is a PhD candidate in the lab of Professor William DeGrado at the University of California, San Francisco. She studies the structure of the influenza M2 proton using lipidic cubic phase crystallization techniques and x-ray diffraction at synchrotron and XFEL sources. The M2 protein is among the smallest proton channels found in nature and is also a drug target against the flu. Her work focuses on the proton conduction mechanism of the M2 channel, particularly the involvement of water in proton transport and the structural characterization of how drugs and novel inhibitors bind to the channel and block proton conduction.
The M2 proton channel of influenza A is a drug target that is essential for replication of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in “wires” inside the channel pore have been proposed to play a role in the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. Previous crystallographic structures determined using a synchrotron radiation source were biased by cryogenic data collection conditions, and room-temperature data collection was limited by radiation damage. These problems have been avoided through room temperature diffraction at an X-ray free electron laser (XFEL).
Data were collected at an XFEL source to a resolution of 1.4 Å at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Here, we examine the ordering of water in the M2 pore within crystals containing only the Copen conformation, which is an intermediate that accumulates at high protonation of the His37 tetrad. This allows us to access multiple protonation states of His37 in the Copen conformation and probe changes in solvent ordering prior to and following the release of a proton into the viral interior.
At pH 5.5, a continuous hydrogen bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at higher pH, where the Copen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.
- "XFEL structures of the M2 proton channel of influenza A reveal pH-dependent water networks under room temperature conditions." Thomaston JL, Woldeyes RA, Nakane T, Yamashita A, Tanaka T, Koiwai K, Brewster AS, Barad BA, Chen Y, Lemmin T, Uervirojnangkoorn M, Arima T, Kobayashi J, Masuda T, Suzuki M, Sugahara M, Sauter NK, Tanaka R, Nureki O, Tono K, Joti Y, Nango E, Iwata S, Yumoto F, Fraser JS, DeGrado WF. In press, PNAS July 2017.
- "Crystal structure of the drug-resistant S31N influenza M2 proton channel." Thomaston JL, DeGrado WF. Protein Science 25(8):1551-4. August 2016.
- "High resolution structures of the M2 proton channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction." Thomaston JL, Alfonso-Prieto M, Woldeyes R, Fraser JS, Klein ML, Fiorin G, DeGrado WF. PNAS 112(46):14260-5. November 2015.
- "Detection of drug-induced conformational change of a transmembrane protein in lipid bilayers using site-directed spin labeling." Thomaston JL, Nguyen P, Brown EC, Upshur MA, Wang J, DeGrado WF, Howard KP. Protein Science 22(1): 65–73. January 2013.
Maharani Pertiwi Koentjoro is a 3rd year PhD student and the Monbukagakusho fellow in the United Graduate School of Agricultural Science, Gifu University, Japan. She holds a BA in Institute of Technology “Sepuluh Nopember”, Indonesia, and a Master in Gadjah Mada University, Indonesia. Her research interests include a molecular biological and biochemical investigation on bacteria. Currently she is working on projects structural studies of complex molecular machines that initiates LysR-Type Transcription Regulator in bacteria.
Cupriavidus necator NH9, which can utilize chlorocatechol as a sole carbon and energy source, degrades chlorocatechol with enzymes of the ortho-cleavage pathway. These enzymes are coded in the cbnABCD operon, of which expression is specifically regulated by a LysR-type transcriptional regulator (LTTR) CbnR. CbnR forms a tetramer and can be regarded as a dimer of dimers. The tetrameric CbnR has four DNA- binding domains and these DNA-binding domains recognize approximately 60 bp DNA sequence. The binding sequence is composed of two binding sites, recognition binding site (RBS) and activation binding site (ABS). Each binding site seems to be recognized by two DNA-binding domains in the tetramer. While the crystal structure of the tetrameric CbnR has already been determined1, the molecular mechanism of DNA recognition by CbnR remains elusive. We therefore initiated the crystal structure analysis of DNA-binding domain of CbnR (CbnR(DBD)) in complex with RBS. The crystal structure would give an insight into the molecular mechanism of the CbnR-DNA interaction, which is the first step to understand the gene activation mechanism by LTTR.
Here we report the crystal structure of CbnR(DBD) (residues 1 - 87) in complex with RBS, a 25-bp DNA fragment. The crystal structure was determined by the MR-native SAD method at 2.55 Å resolution with Rwork/Rfree of 0.221/0.264. The crystal structure shows that dimeric CbnR(DBD) interacts with RBS. The dimeric CbnR(DBD) adopts essentially the same conformation as that in the tetramic CbnR with the root mean squares deviation of 1.1 Å (174 Cα atoms). The a3 helix and the winged region of the winged-helix turn helix (wHTH) motif in CbnR(DBD) directly interact with the major and minor grooves of promoter sequence, respectively, and the interactions seem to bend DNA by approximately 30°. To further analyse the molecular mechanism of their interaction, biochemical analysis is in progress.
Muraoka, S., Okumura, R.., Uragami, Y., Nonaka, T., Ogawa, N., Miyashita, K. and Senda, T (2003) Purification and crystallization of a Lysr–type transcriptional regulator CbnR from Ralstonia eutropha NH9. Protein and Peptide Letters, 10 (3): 325–329.
Ogawa, N. McFall, SM., Klem, TJ., Miyashita, K and Chakrabarty, AM (1999) Transcriptional activation of the clorocatechol degradative genes of Ralstonia eutropha NH9. Journal of Bacteriology, 181 (21): 6697–6705.
Ruangprasert, A., Craven, SH., Neidle, EL., and Momany, C. 2010. Full–length structures of BenM and two variants reveal different oligomerization schemes for LysR–Type transcriptional regulators. Journal of Molecular Biology, 404:568–586.
Devese, L., Smirnova, I., Lonneborg, R., Kapp, U., Brzezinski, P., Leonard, GA. and Dian, C (2011) Crystal structures of DntR inducer binding domains in complex with salicylate offer insights into the activation of LysR–type transcriptional regulators. Molecular Microbiology, 81 (2): 354–367.
Alanazi, AM., Neidle, EL., and Momany, C (2013) The DNA-binding domain of BenM reveals the structural basis for the recognition of a T-N11-A sequence motif by LysR-type transcriptional regulators. Acta Crystallographica, D69: 1995-2007.
Paul Scherrer Institute, Switzerland
Nadia Opara joined the CINA group at the Biozentrum University of Basel and the LMN at PSI in Switzerland in 2014 in the frame of SNI PhD school program to work on a project aiming at improving sample preparation methods for XFEL-based protein nano-crystallography. Beforehand she completed her bachelor studies in chemistry and a master program in molecular biotechnology at the Lodz University of Technology in Poland.
Classical crystallography methods based on synchrotrons usually require crystals of relatively large dimensions, i.e. above about 5 micrometers. The recent availability of X-ray free electron laser sources (XFELs) providing femtosecond X-ray pulses of ultrahigh brightness facilitate the investigation of nanocrystals. However, in this case data collection has to be performed in the mode of the serial crystallography in so-called diffraction-before-destruction regime because the probed area of the sample is completely destroyed after the interaction with ultraintense radiation. As thousands of crystals have to be provided sequentially to the XFEL beam, selection of an efficient sample delivery system is crucial to minimize protein consumption during data collection.
Delivery methods applied so far include steady streaming liquid jets [1,2] of the crystal suspension. The application of more viscous media like lipidic cubic phase (LCP) , agarose  or hyaluronic acid  matrices has also been demonstrated. However all these methods use significant amounts of the precious protein, which cannot be recovered even if not directly probed.
Recent developments of drop of demand methods  or fixed targets [1,7] allow overcoming this problem. But still, handling of the fragile crystals should be gentle or, at best, avoided.
Microfabricated silicon chips with ultrathin Si3N4 membranes provide the possibility to regularly position crystals on precisely defined spots by direct crystallization using classical vapor diffusion method . The sample consumption is minimal since crystal growth takes place in nanoliter volume cavities. No additional sample transfer is needed, because X-rays are probing the crystals at the spot where they grew on the X-ray-transparent ultrathin amorphous silicon nitride membranes. Assembly with a second chip to form a hermetically sealed sandwich protects specimens from dehydration and facilitates in situ diffraction data collection at room temperature, as demonstrated in a synchrotron experiment providing high-resolution patterns [Fig. 1].
- Muniyappan S et al. (2015) Recent advances and future prospects of serial crystallography using XFEL and synchrotron X-ray sources. Bio Design 3:2
- Steinke I et al. (2016) A liquid jet setup for x-ray scattering experiments on complex liquids at free-electron laser sources. Rev Sci Instrum. 87:063905
- Weierstall U et al. (2014) Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nat Commun. 5:3309
- Conrad CE et al. (2015) A novel inert crystal delivery medium for serial femtosecond crystallography. IUCrJ 2:421-30
- Sugahara M et al. (2016) Oil-free hyaluronic acid matrix for serial femtosecond crystallography. Sci Rep. 6:24484
Inha University, South Korea
Ji Won Kim has her passion in improving the health and wellbeing for head and neck cancer patients. Also she has an interest in salivary gland disease and aging. She is working in Inha university medical center after ENT resident training and fellowship in Asan Medical center. She worked as a member in asan institute for life science. She has a lot of papers about head and neck cancers treatment and therapeutic approach to overcome salivary dysfunction.
Backgound: We suggested a novel strategy termed induced phenotype targeted therapy (IPTT) using radiation. The aim of this study is to expand the the clinical implications of IPTT including metastatic tumors. We investigated the candidate drugs such as checkpoint kinase 1 (Chk 1) inhibitor to induce phenotype caspase-3 and found the mechanism of activating the prodrug.
Methodology & Theoretical Orientation: We designed a caspase-3 specific activatable prodrug, DEVD-S-DOX, containing doxorubicin linked to a peptide moiety (DEVD) cleavable by caspase-3 upon apoptosis. To induce apoptosis systemically in the metastatic tumor, we used a Chk 1 inhibitor, LY2603618. clonogenicity, cell cycle distribution and apoptosis were assessed in breast cancer cell lines with prodrug and LY2603618, seperatively, and combination.The in vivo antitumor activity of the caspase-3-specific activatable prodrug combined with LY2603618 was investigated in C3H/HeN tumor-bearing mice (n = 5 per group) and analyzed with the Student's t test or Mann-Whitney U test. All statistical tests were two-sided. We confirmed the basic principle using a caspase-sensitive nanoprobe (Apo-NP).
Findings: The in vitro studies showed that the cell cycle arrest of p53-deficient cancer cells by Chk1 inhibitor, thus leading to facilitated caspase-3 upon apoptosis. The growth of MDA-MB-231, which is p53-deficient, was effectively suppressed by daily oral administration of LY2603618 when treated with DEVD-S-DOX in vivo without evident toxicities.
Conclusion & Significance: The combination therapy using DEVD-S-DOX and Chk1 inhibitor could effectively treat p53-deficient breast cancer by selectively chemosensitizing cancer cells with low toxic adverse effects.
- Lee BS, Cho YW, Kim GC, Lee DH, Kim CJ, Kil HS, Chi DY, Byun Y, Yuk SH, Kim K, Kim IS, Kwon IC, Kim SY. Induced phenotype targeted therapy: radiation-induced apoptosis-targeted chemotherapy. J Natl Cancer Inst. 2014 Dec 12;107(2).
- Chung SW, Choi JU, Lee BS, Byun J, Jeon OC, Kim SW, Kim IS, Kim SY, Byun Y. Albumin-binding caspase-cleavable prodrug that is selectively activated in radiation exposed local tumor. Biomaterials. 2016 Jul;94:1-8.
- Chung SW, Kim GC, Kweon S, Lee H, Chang HW, Kim JW, Son WC, Kim SY, Byun Y. Metronomic oral doxorubicin with combination of Chk1 inhibitor for the treatment of p53-deficient breast cancer. Mol Cancer Ther.submitted
Stephanie Morais performs her pHD under Tatiana Souza supervision and coordinate projects involving advances in leukemia treatment advances. She is part of the team since 2013 and has expertise not only in molecular and structural biology but also in cancer biology.
Acute Lymphoid Leukemia (ALL) is the most common neoplasia in childhood. The multi-therapeutic treatment resulted in remarkable advances in treatment of children, with 90.4% survival rate. L-asparaginase has been a central component of ALL therapy for over 40 years and acts by depleting plasma asparagine. In contrast to the normal cells, tumor cells lack the ability to synthesize asparagine and thus depend on external uptake of this amino acid for growth. Nowadays, three asparaginases are used in therapy: native L-asparaginase II from Escherichia coli, a pegylated form of this enzyme and L-asparaginase isolated from Erwinia chrysanthemi. Among the commercially available L-asparaginases, the E. coli enzyme presents the highest catalytic activity but also the highest toxicity, due to its further ability to hydrolyze glutamine, generating glutamate. Moreover, the immune response in patients under therapy with bacterial asparaginases can result in enzyme neutralization and the need to proceed the treatment with one of the alternative L-asparaginases. Based on the analysis of the available crystal structures we have designed, produced and crystallized E. coli asparaginase with modifications. Crystals diffracted up to 1.65 Å resolution at the Soleil Synchrotron. We combine structural analysis with kinetic and cellular approaches in order to identify the determinants of E. coli asparaginase toxicity. In addition, we have been working on the production of modified human asparaginases for structural characterization, kinetic and anti-leukemic activity assays. The introduction of human asparaginase in ALL treatment would avoid the problems caused by the bacterial enzymes, however a major difficulty in the therapeutic use of the human enzyme comes from the fact that human asparaginases need to undergo activation through an auto-cleavage step, which was shown to be a low efficiency process in vitro, reducing the enzyme activity. These structural analysis gather insights about how engineering asparaginases can improve ALL treatment.
- MORAIS, S. B. ; WEILER, A. V. P. ; MURAKAMI, M. T. ; Souza, T. A. C. B. Characterization of Alanine Aminotransferase from Trypanosoma cruzi. Protein & Peptide Letters, 2016
- SANTOS, FRED LUCIANO NEVES ; CELEDON, PAOLA ALEJANDRA FIORANI ; ZANCHIN, NILSON IVO TONIN ; Brasil, tatiana de arruda campos ; FOTI, LEONARDO ; SOUZA, WAYNER VIEIRA DE ; SILVA, EDMILSON DOMINGOS ; GOMES, YARA DE MIRANDA ; KRIEGER, Marco Aurélio . Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease. Plos One, v. 11, p. e0161100, 2016.
- SANTANA, ALINE GUIMARÃES ; GRACHER, ANA HELENA PEREIRA ; RÜDIGER, ANDRÉ LUIS ; ZANCHIN, NILSON IVO TONIN ; CARVALHO, PAULO COSTA ; CIPRIANI, THALES RICARDO ; DE ARRUDA CAMPOS BRASIL DE SOUZA, TATIANA . Identification of potential targets for an anticoagulant pectin. Journal of Proteomics (Print), v. 1, p. 1-4, 2016.
- MORAES, EDUARDO ; MEIRELLES, GABRIELA ; HONORATO, RODRIGO ; DE SOUZA, Tatiana ; DE SOUZA, EDMARCIA ; MURAKAMI, MARIO ; DE OLIVEIRA, PAULO ; Kobarg, Jörg . Kinase Inhibitor Profile for Human Nek1, Nek6, and Nek7 and Analysis of the Structural Basis for Inhibitor Specificity. Molecules (Basel. Online), v. 20, p. 1176-1191, 2015.
- DOMINGUES, MARIANE NORONHA ; SFORÇA, MAURICIO LUIS ; SOPRANO, ADRIANA SANTOS ; LEE, JACK ; DE SOUZA, TATIANA DE ARRUDA CAMPOS BRASIL ; CASSAGO, ALEXANDRE ; PORTUGAL, RODRIGO VILLARES ; DE MATTOS ZERI, ANA CAROLINA ; MURAKAMI, MARIO TYAGO ; SADANANDOM, ARI ; DE OLIVEIRA, PAULO SERGIO LOPES ; BENEDETTI, CELSO EDUARDO . Structure and Mechanism of Dimer-Monomer Transition of a Plant Poly(A)-Binding Protein upon RNA Interaction: Insights into Its Poly(A) Tail Assembly. Journal of Molecular Biology, v. 427, p. 2491-2506, 2015.