Titel Deelnemers "Korte inhoud" "An integrative structural biology approach in the study of bacterial persistence and virulence" "Yurong Wen" "Antibiotics have provided an enormous contribution to humansU+2019 fight against infectious microorganisms resulting in a dramatic decrease in mortality due to microbial infections around the globe. However, the widespread and uncontrolled use of antibiotics, particularly as growth promoters in feedstock caused an environmental stress for bacterial evolution and Multi Drug Resistance (MDR) and Multi Drug Tolerance (MDT) are two of the major challenges for further eradication of the bacterial infections. In recent years, this antibiotics crisis has become one of the most serious public health concerns and threats by renewed awareness. Therefore, how the bacteria could survive under the situation of antimicrobial treatment and other environmental stress, and how the bacteria infect the host organism are essential to develop novel antibacterial strategies. Finding the right strategies such as discovery of new antimicrobial drugs are urgent to fight with the current crisis of antibiotic therapy. In this PhD research, we picked up two major process concerning bacterial infections which are bacterial persistence and virulence respectively. Among others, we investigated Toxin-antitoxin systems of which the recent discovery of new families is one of the most exciting molecular microbial phenomena crucial to understand the bacterial behavior. Toxin-antitoxin systems are widespread two or three component systems encoded in the bacterial genome and highlighted to be involved in the persistence, biofilm formation, pathogenicity, programmed cell death and many other cellular processes in bacteria. Due to those characteristics, it also has potential as attractive targets in antimicrobial drug discovery. We applied multiple structural biology, biochemical and biophysical techniques to explore the molecular mechanism of the HipAB toxin-antitoxin system coded in S.oneidensis MR-1 genome, involved in biofilm and persistence. Multiple atomic structures including individual HipB as well as complexes of HipA with non-hydrolysable ATP analogues, Mg2+ and HipA-HipB with its operator DNA were solved. We showed that phosphorylated HipA can engage in an unexpected ternary complex with HipB and double stranded operator DNA that is distinct from the prototypical counterpart complex from E.coli. The structure of HipB in complex with its operator DNA reveals a flexible C-terminus that is sequestering HipA in the ternary complex, indicative of its role in binding HipA to abolish its function in persistence. The structure of a HipA mutant devoid of phosphorylation capacity in complex with a non-hydrolyzable ATP analogue uncovers that HipA autophosphorylation is coupled to an unusual conformational collapse of its pLoop. However, HipA is unable to phosphorylate the translation factor Elongation factor Tu (EF-Tu), contrary to previous reports but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought. Aside from the investigation of bacterial persistence, we also study the bacterial secretion systems which play a central role in modulating the interactions between the bacteria and its host organisms or extracellular environment by secreting diverse virulence factors. We focused on the opportunistic human pathogen Pseudomonas aeruginosa type 2 secretion system which is one of the most versatile systems used in gram negative bacteria. In P.aeruginosa, the T2SS machinery contains 12 protein subunits which can be distinguished into 5 subassemblies. Among those 12 protein subunits, XcpP is thought to be tightly involved in a procedure that bridges the inner membrane and the outer membrane secretin XcpQ; the assembly of XcpP and XcpQ is crucial to understand the transport machinery. We carried out multiple mass spectrometry experiments and tightly collaborated with a PhD student from Prof. Savvas SavvidesU+2019 group, Ruben Van der Meeren focusing on the biophysical, biochemical and functional analysis of the assembly between XcpP and XcpQ. We demonstrated that the P.aeruginosa T2SS secretin XcpQ periplasmic domain is a dimeric protein which oligomerize into a functional dodecameric assembly with C6 symmetry instead of the previous reported dodecameric assembly with C12 symmetry. We proposed the two transition states for XcpP and XcpQ periplasmic interaction which consist of the U+201CopenU+201D and U+201CclosedU+201D state of the Type 2 secretion system to transport the exoprotein from the periplasm to the extracellular milieu. Overall, we have successfully applied mass spectrometric based integrative structural biology to study the bacterial persistence and virulence. The approach combining the biochemical, structural biological, biophysical techniques and especially the mass spectrometric based techniques that could bridge those techniques are powerful for the structural and functional investigation of supermolecular assembly." "WeNMR: Structural Biology on the Grid" "Tsjerk Wassenaar, Mark Van Dijk, Nuno Loureiro-Ferreira, Gijs Van Der Schot, Sjoerd De Vries, Christophe Schmitz, Johan Van Der Zwan, Rolf Boelens, Andrea Giachetti, Lucio Ferella, Antonio Rosato, Ivano Bertini, Torsten Herrmann, Hendrik Jonker, Anurag Bagaria, Victor Jaravine, Peter Güntert, Harald Schwalbe, Wim Vranken, Jurgen Doreleijers, Gert Vriend, Geerten Vuister, Daniel Franke, Alexey Kikhney, Dmitri Svergun, Rasmus Fogh, John Ionides, Ernest Laue, Chris Spronk, Simon Jurksa, Marco Verlato, Simone Badoer, Stefano Dal Pra, Mirco Mazzucato, Eric Frizziero, Alexandre Bonvin" "The WeNMR (http://www.wenmr.eu) project is a European Union funded international effort to streamline and automate analysis of Nuclear Magnetic Resonance (NMR) and Small Angle X-Ray scattering (SAXS) imaging data for atomic and near-atomic resolution molecular structures. Conventional calculation of structure requires the use of various software packages, considerable user expertise and ample computational resources. To facilitate the use of NMR spectroscopy and SAXS in life sciences the WeNMR consortium has established standard computational workflows and services through easy-to-use web interfaces, while still retaining sufficient flexibility to handle more specific requests. Thus far, a number of programs often used in structural biology have been made available through application portals. The implementation of these services, in particular the distribution of calculations to a Grid computing infrastructure, involves a novel mechanism for submission and handling of jobs that is independent of the type of job being run. With over 450 registered users (September 2012), WeNMR is currently the largest Virtual Organization (VO) in life sciences. With its large and worldwide user community, WeNMR has become the first Virtual Research Community officially recognized by the European Grid Infrastructure (EGI)." "Native ion mobility-mass spectrometry and related methods in structural biology" "Albert Konijnenberg, Annika Kotter (geb. Butterer)" "Mass spectrometry-based methods have become increasingly important in structural biology - in particular for large and dynamic, even heterogeneous assemblies of biomolecules. Native electrospray ionization coupled to ion mobility-mass spectrometry provides access to stoichiometry, size and architecture of noncovalent assemblies; while non-native approaches such as covalent labeling and H/D exchange can highlight dynamic details of protein structures and capture intermediate states. In this overview article we will describe these methods and highlight some recent applications for proteins and protein complexes, with particular emphasis on native MS analysis. This article is part of a Special Issue entitled: Mass spectrometry in structural biology. (C) 2012 Elsevier B.V. All rights reserved." "Bacterial Membranes: Structural and Molecular Biology" "Membranes are pivotal components of life acting as formidable insulators that demarcate a living cell, generate energy in the form of ion gradients, transport ions, proteins, nucleic acids, nutrients and metabolites, and provide transduction systems to sense the environment and to communicate with other cells. Membranes also provide shape and structure to cells and are important in cell motility. In addition they fulfil a scaffolding function for proteins and organelles that interact with the extracellular environment.Written by specialists in the field, this book provides a comprehensive overview of the structural and molecular biology of cellular processes that occur at or near bacterial membranes. The authors present and discuss recent progress on the function and involvement of membranes in bacterial physiology enabling a greater understanding of the molecular details of the cell envelope, its biogenesis and function. Topics covered include: cell wall growth, shape and division, the outer membrane of Gram-negative bacteria, outer membrane protein biosynthesis, bacterial lipoproteins, mycobacteria, lipid composition, ABC transporters, transport across the outer membrane, drug passage across membranes, bacterial membrane proteins, secretion systems, signal transduction, signalling mechanisms, bacterial membranes in adhesion and pathogenesis, and membranes as a drug target.This cutting-edge text will provide a valuable resource for all those working in this field and is recommended for all microbiology libraries." "A general protocol for the generation of Nanobodies for structural Biology" "Els Pardon, Toon Laeremans, Sarah Triest, Armin Ruf, Serge Muyldermans, Wim G. J. Hol, Brian K Kobilka, Jan Steyaert" "There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technology has a competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo-matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography in a time span of 6-12 months." "Structural and biochemical evaluation of natural and modified Nucleic Acids and their interactions with drugs and biomolecules for therapeutic and synthetic biology application" "Mohitosh Maiti" "Title: ""Structural and biochemical evaluation of natural and modified Nucleic Acids and their interactions with drugs and biomolecules for th erapeutic and synthetic biology application"" Deoxyribo and ribo nucleic acids (DNA and RNA) are the two key players i nvolved in the central dogma of molecular biology (DNA to RNA&nbs p;to Protein). Generally, DNA acts as the storage of genetic information while RNA molecules for long time are being considered as the intermedi ates to transfer the genetic information from DNA to the functional prot ein world. However, this notion regarding RNA is significantly altered b y some of the recent decades discoveries, where RNAs are shown to have catalytic function (by ribozymes) and a wide regulatory role in gene-exp ression by endogenous non-coding RNAs like microRNA (miRNA) and by short interfering RNA (siRNA) mediated RNA interference (RNAi). This in turn has provided us the novel opportunities to target functionally deregulat ed RNAs involved in pathogenesis. Targeting the RNA level in some diseas es can be advantageous over protein targets where there is a lack of str ucturally drugable proteins. On the other hand, targeting at the DNA lev el is difficult since nuclear membrane and highly packed DNA provide a b arrier for the access to the drug molecules. Targeting in the RNA level can be achieved either by small molecular approach or by using modified oligonucleotides (e.g. antisense agent, modified-siRNA/miRNA etc). Each approach has its inherent merits and limitations as well.Natural RNA adopts diverse secondary and tertiary structures, and possesses necessa ry structural flexibility that is crucially important for its biological function. Structural understanding of the RNA and its interactions with other RNAs, biomolecules and small molecule ligands is necessary for th e understanding of their biological function and for the design of suita ble drugs that target RNA. In the case of RNA targeting by modified olig onucleotides, they should mimic the natural recognition principles and t herefore their three-dimensional structure has to be compatible with the existing natural RNA and protein machinery. Beside the therapeutic appl ication of chemically modified oligonucleotides, recent studies demonstr ate a growing interest in chemically modified nucleic acids for their po ssible application in synthetic biology. These oligonucleotides should b e structurally and biochemically orthogonal to the natural nucleic acids but should be able to carry out storage and transfer of genetic informa tion independently inside a living cell. Here as well the structural and biophysical studies of chemically engineered alternative information sy stems and their comparison with the natural DNA/RNA are necessary for th e selection of suitable candidates for further biological evaluation. Th e comparative study can also answer the questions related to why nature has selected DNA/RNA as the genetic information systems rather than an y other nucleic acids.The second chapter (first project) of this the sis describes the identification and characterization of secondary struc tural forms of mature miRNAs. miRNAs are a class of endogenous, short, non-coding RNAs that post-transcriptionally regulate gene-expression by b inding with partially complementary sequences in mRNA and thereby steric ally blocking translation or by triggering mRNA degradation. An equilibr ium between hairpin and homo-duplex form of the hsa-mir-520h mature miRN A strand has been characterized by using solution state 1D and 2D proton NMR spectroscopy. A detailed sequence analysis was performed for all mi RNAs from the database of known miRNAs (miRBase). The statistical analys is of the resulting folding and alignment data revealed the potential of a large number of miRNAs to form significant self-complementary hairpin and/or homo-duplex structures in solution under physiological condition s. Several mechanistic models are discussed by which self-complementarit y of mature miRNAs can provide a mechanistic tuning and a regulatory sop histication to the process of miRNA mediated gene regulation.The thi rd chapter deals with RNA-targeting by small-molecular approach for a ne wly emerging therapeutic RNA target, pre-microRNA. Over-expressions of m iRNAs are being increasingly linked with many diseases including differe nt types of cancer. In this study, the role of some known small molecula r therapeutics has been investigated for their ability to bind with a pr e-miRNA (hsa-mir-155) and thereby to interfere with the final step of mi RNA biogenesis, in which an RNase III enzyme Dicer catalyzes the formati on of mature miRNA from the corresponding inactive stem-loop hairpin (pr e-miRNA) substrate. Potential binding and inhibition effects have been d emonstrated by some of these analogues. They can be used as leads for fu rther development of potent and selective small molecular miRNA-antagoni sts.In the fourth chapter structure determination of several altrito l nucleic acids (ANA)-modified siRNAs was performed and binding of sever al ANA and thymidine residue (dTdT)-modified siRNAs were investigated wi th the PAZ domain of Argonaute 2 protein of Drosophila melanogaster. ANA contains six-membered altritol sugar in the sugar-phosphate backbone. A NA-modified siRNA (ANA/RNA) duplexes adopt a geometry very similar to th e naturally occurring A-type siRNA duplex. The six-membered altritol sug ar in ANA occurs in a chair conformation with the nucleobase in an axial position. Binding studies demonstrate that modifications in the double- stranded region of the antisense strand have some small effects on the b inding affinity. Modification of the 3′ overhang (unpaired protrud ing part of a DNA/RNA duplex) with dTdT shows a six-fold increase in the binding affinity compared with the unmodified siRNA, whereas modificati on of the 3′ overhang with ANA largely decreases the binding affin ity. The structural and binding data of this study in combination with t he previous results of biological (RNAi) activity shed light on the unde rstanding of the therapeutic potential of chemically modified siRNAs (as RNA-targeting tools) from a structure-function point of view, and highl ight for the future therapeutic design of chimeric modified siRNAs with improved protein recognition and RNAi activity. The fifth chapter describes the structural and biochemical evaluation of de oxyxylonucleic acid (dXNA) as an orthogonal genetic information system f or synthetic biology application. Solution structure and conformational analysis of two self-complementary, fully modified dXNA oligonucleotides are presented, as determined by CD and NMR spectroscopy. These studies are the initial experimental proof of the structural orthogonality of dX NAs. In aqueous solution, dXNA duplexes predominantly form a linear ladd er-like (type-1) structure, which is significantly different from natura l DNA/RNA duplex structure. The corresponding nucleoside triphosphates ( dXNTPs) were synthesized and evaluated for their ability to be incorpora ted into a growing DNA chain by using several natural and mutant DNA pol ymerases. Despite the structural orthogonality of dXNA, DNA polymerase&n bsp;ß mutant is able to incorporate the dXNTPs, showing initial DNA -dependent dXNA polymerase activity." "Integrative structural biology studies of HIV-1 reverse transcriptase binding to a high-affinity DNA aptamer" "Kalyan Das" "The high-resolution crystal structure of HIV-1 reverse transcriptase (RT) bound to a 38-mer DNA hairpin aptamer with low pM affinity was previously described. The high-affinity binding aptamer contained 2′-O-methyl modifications and a seven base-pair GC-rich tract and the structure of the RT-aptamer complex revealed specific contacts between RT and the template strand of the aptamer. Similar to all crystal structures of RT bound to nucleic acid template-primers, the aptamer bound RT with a bend in the duplex DNA. To understand the structural basis for the ultra-high-affinity aptamer binding, an integrative structural biology approach was used. Hydrogen-deuterium exchange coupled to liquid chromatography-mass spectrometry (HDX-MS) was used to examine the structural dynamics of RT alone and in the presence of the DNA aptamer. RT was selectively labeled with 15N to unambiguously identify peptides from each subunit. HDX of unliganded RT shows a mostly stable core. The p66 fingers and thumb subdomains, and the RNase H domain are relatively dynamic. HDX indicates that both the aptamer and a scrambled version significantly stabilize regions of RT that are dynamic in the absence of DNA. No substantial differences in RT dynamics are observed between aptamer and scrambled aptamer binding, despite a large difference in binding affinity. Small-angle X-ray scattering and circular dichroism spectroscopy were used to investigate the aptamer conformation in solution and revealed a pre-bent DNA that possesses both A- and B-form helical character. Both the 2′-O-methyl modifications and the GC tract appear to contribute to an energetically favorable conformation for binding to RT that contributes to the aptamer’s ultra-high affinity for RT. The X-ray structure of RT with an RNA/DNA version of the aptamer at 2.8 Å resolution revealed a potential role of the hairpin positioning in affinity. Together, the data suggest that both the 2′-O-methyl modifications and the GC tract contribute to an energetically favorable conformation for high-affinity binding to RT." "Computational strategies and challenges for using native ion mobility mass spectrometry in biophysics and structural biology" "Timothy M. Allison, Perdita Barran, Sarah Cianferani, Matteo T. Degiacomi, Valeprimerie Gabelica, Rita Grandori, Erik G. Marklund, Thomas Menneteau, Lukasz G. Migas, Argyris Politis, Michal Sharon, Frank Sobott, Konstantinos Thalassinos, Justin L. P. Benesch" "Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase noncovalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods have improved, there have been accompanying developments in computational approaches for analyzing and exploiting the profusion of MS data in a structural and biophysical context. In this perspective, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed. Our perspective is based on discussions within the European Cooperation in Science and Technology Action on Native Mass Spectrometry and Related Methods for Structural Biology (EU COST Action BM1403), which involved participants from across Europe and North America. It is intended not as an in-depth review but instead to provide an accessible introduction to and overview of the topic-to inform newcomers to the field and stimulate discussions in the community about addressing existing challenges. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05792) focuses on software tools available to help researchers tackle some of the challenges enumerated here." "Understanding nuclear receptor form and function using structural biology" "Fraydoon Rastinejad" "Nuclear receptors (NRs) are a major transcription factor family whose members selectively bind small-molecule lipophilic ligands and transduce those signals into specific changes in gene programs. For over two decades, structural biology efforts were focused exclusively on the individual ligand-binding domains (LBDs) or DNA-binding domains of NRs. These analyses revealed the basis for both ligand and DNA binding and also revealed receptor conformations representing both the activated and repressed states. Additionally, crystallographic studies explained how NR LBD surfaces recognize discrete portions of transcriptional coregulators. The many structural snapshots of LBDs have also guided the development of synthetic ligands with therapeutic potential. Yet, the exclusive structural focus on isolated NR domains has made it difficult to conceptualize how all the NR polypeptide segments are coordinated physically and functionally in the context of receptor quaternary architectures. Newly emerged crystal structures of the peroxisome proliferator-activated receptor-γ-retinoid X receptor α (PPARγ-RXRα) heterodimer and hepatocyte nuclear factor (HNF)-4α homodimer have recently revealed the higher order organizations of these receptor complexes on DNA, as well as the complexity and uniqueness of their domain-domain interfaces. These emerging structural advances promise to better explain how signals in one domain can be allosterically transmitted to distal receptor domains, also providing much better frameworks for guiding future drug discovery efforts." "Structural biology in drug development" "Eveline Lescrinier" "Structural biologists focus on the structure of biomolecules. Several techniques are available to study the structure adopted by a biomolecule and unravel how this structure is related to its constitution and function. For their biological role in a functioning cell or organism, biomolecules interact with each other and/or rather small molecules in their environment. Drugs can exploit interaction with biomolecules to manipulate their biological function to obtain a therapeutic effect. Structure determination of biomolecules that (could) serve as therapeutic target is an important starting point in rational drug design. Once the structure of a biological target is known, a potential binding site for drugs and possible interactions at this site have to be identified. In the stage of drug design this information is a valuable input for modeling experiments. They can virtually scan libraries of compounds by docking them into the binding site. This strategy ranks potential ligands that can be chemically modified to optimize their interaction at the binding site ('lead optimization') in order to improve affinity and selectivity for the biomolecular target. Modeling can also be used to virtually 'build' new molecules starting from possible interactions and shape of the target binding site ('de novo design'). Structural biology can contribute in different stages of drug development to direct the process or optimize existing compounds."