Advances that facilitate the study of large RNA structure and dynamics by nuclear magnetic resonance spectroscopy

The characterization of functional yet nonprotein coding (nc) RNAs has expanded the role of RNA in the cell from a passive player in the central dogma of molecular biology to an active regulator of gene expression. The misregulation of ncRNA function has been linked with a variety of diseases and disorders ranging from cancers to neurodegeneration. However, a detailed molecular understanding of how ncRNAs function has been limited; due, in part, to the difficulties associated with obtaining high‐resolution structures of large RNAs. Tertiary structure determination of RNA as a whole is hampered by various technical challenges, all of which are exacerbated as the size of the RNA increases. Namely, RNAs tend to be highly flexible and dynamic molecules, which are difficult to crystallize. Biomolecular nuclear magnetic resonance (NMR) spectroscopy offers a viable alternative to determining the structure of large RNA molecules that do not readily crystallize, but is itself hindered by some technical limitations. Recently, a series of advancements have allowed the biomolecular NMR field to overcome, at least in part, some of these limitations. These advances include improvements in sample preparation strategies as well as methodological improvements. Together, these innovations pave the way for the study of ever larger RNA molecules that have important biological function.This article is categorized under:RNA Structure and Dynamics > RNA Structure, Dynamics, and ChemistryRegulatory RNAs/RNAi/Riboswitches > Regulatory RNAsRNA Structure and Dynamics > Influence of RNA Structure in Biological SystemsOverview of important sample preparation and methodological advancements that facilitate the study of large RNA structure and dynamics by nuclear magnetic resonance spectroscopy. These innovations pave the way for the study of previously intractable systems.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151321/1/wrna1541.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151321/2/wrna1541_am.pd

Structures of biomolecular complexes by combination of NMR and cryoEM methods

CryoEM is presently providing structures of biocomplexes considered intractable to analysis by other structural techniques. NMR is playing an important role in delivering structural information on dynamics events and conformational heterogeneity. Impressive results were obtained by combining cryoEM and either liquid- or solid-state NMR, revealing the structures of cellular machines, filaments and amyloid fibrils. NMR solution structures of proteins and nucleic acids were fitted, together with crystallographic structures, into cryoEM maps of large complexes, to decipher their assembly mechanisms and describe their functional dynamics. Modelling based on solid-state NMR and cryoEM data provided 3D structure of filaments and fibrils. These NMR approaches validated, but also corrected, atomic models built de novo in cryoEM maps, and provided new structural data on flexible or structurally heterogeneous systems. Combination of cryoEM and NMR became an established hybrid approach in structural biology that significantly contributes to our understanding of functional mechanisms in supramolecular assemblies

Structure Characterization of the 70S-BipA Complex Using Novel Methods of Single-Particle Cryo-Electron Microscopy

Diseases caused by pathogenic bacteria continue to be major health concerns. For example, it is estimated that in the year 2000 typhoid fever caused over 21,000,000 illnesses and ~200,000 deaths (Crump et al., 2004). The disease is caused by S. typhi, a closely-related serotype of S. typhiumurium, the salmonella strain in which BipA was first identified. The CDC estimated that in 2013, multidrug resistant bacteria caused over 2 million infections in the United States, ending in more than 23,000 deaths (CDC, 2013). This number is set to rise as more bacteria become resilient to the collection of conventional antibiotics. The increasing number of multidrug resistant bacterial strains necessitates the development of new antimicrobial drugs. BipA is an attractive target for drug research. As mentioned in Section 2.5.2, BipA is ubiquitous in eubacteria and lower eukaryotes such as protozoa, but is absent from higher-order eukaryotes such as humans. Because the protein is essential for bacterial survival, BipA presents a major vulnerability of pathogenic bacteria. A drug targeting the protein itself or its interactions to the ribosome will disable only the bacteria, but have no effect on the eukaryotic host. A comprehensive model of BipA bound to the 70S ribosome will provide unparalleled insight into BipA's binding site and its mechanism. Toward this goal, cryo-EM techniques were employed to visualize the binding site of BipA on the 70S ribosome, characterize its interactions with the ribosome, and elucidate its mechanism on the ribosome. An X-ray structure of isolated BipA-GMPPNP was elucidated, by collaborators, and used for further molecular modeling of the protein to reveal possible atomic interactions between BipA and 70S ribosome. Additional biochemical studies were performed to fully characterize the specific ribosomal complex that optimizes binding of the factor. Together, the cryo-EM reconstruction, the BipA X-ray structure, the subsequent molecular modeling, and the additional biochemical studies provide a comprehensive model for BipA binding. Over the last years, the introduction of new automated algorithms for particle selection (AutoPicker) and classification (RELION) for the cryo-EM technique has revolutionized the workflow of the entire imaging and reconstruction process. The BipA dataset was primed to be used as a test bed for these algorithms and classification technique, respectively. Using old and new techniques to process the dataset allows a discussion of how the single particle reconstruction process can be vastly improved, with greater automation and efficiency

Structure-Function insights of Jaburetox and Soyuretox: Novel intrinsically disordered polypeptides derived from plant ureases

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) do not have a stable 3D structure but still have important biological activities. Jaburetox is a recombinant peptide derived from the jack bean (Canavalia ensiformis) urease and presents entomotoxic and antimicrobial actions. The structure of Jaburetox was elucidated using nuclear magnetic resonance which reveals it is an IDP with small amounts of secondary structure. Different approaches have demonstrated that Jaburetox acquires certain folding upon interaction with lipid membranes, a characteristic commonly found in other IDPs and usually important for their biological functions. Soyuretox, a recombinant peptide derived from the soybean (Glycine max) ubiquitous urease and homologous to Jaburetox, was also characterized for its biological activities and structural properties. Soyuretox is also an IDP, presenting more secondary structure in comparison with Jaburetox and similar entomotoxic and fungitoxic effects. Moreover, Soyuretox was found to be nontoxic to zebra fish, while Jaburetox was innocuous to mice and rats. This profile of toxicity affecting detrimental species without damaging mammals or the environment qualified them to be used in biotechnological applications. Both peptides were employed to develop transgenic crops and these plants were active against insects and nematodes, unveiling their immense potentiality for field applications.Fil: Grahl, Matheus V. Coste. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Lopes, Fernanda Cortez. Universidade Federal do Rio Grande do Sul; BrasilFil: Martinelli, Anne H. Souza. Universidade Federal do Rio Grande do Sul; BrasilFil: Carlini, Célia Regina R. S.. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Three-dimensional reconstruction of macromolecular assemblies using transmission electron microscopy and 3D reconstruction softwares

Master'sMASTER OF SCIENC

Protein Structure

Since the dawn of recorded history, and probably even before, men and women have been grasping at the mechanisms by which they themselves exist. Only relatively recently, did this grasp yield anything of substance, and only within the last several decades did the proteins play a pivotal role in this existence. In this expose on the topic of protein structure some of the current issues in this scientific field are discussed. The aim is that a non-expert can gain some appreciation for the intricacies involved, and in the current state of affairs. The expert meanwhile, we hope, can gain a deeper understanding of the topic

The architecture of polyketide synthases

Since the discovery of penicillin over a century ago, secondary metabolites from all kingdoms of life have proven to be of high medical value. One class of proteins prevalent in the production of secondary metabolites are polyketide synthases (PKSs). Their polyketide products are complex organic compounds based on carbon chains assembled from carboxylic acid precursors. Many polyketides are produced by their hosts with the primary purpose of gaining an advantage in their ecological niche. To contribute to such an advantage, a significant proportion of polyketides are active against pro- and eukaryotic microorganisms. Type I PKSs are giant multienzyme proteins employing an assembly line logic for the synthesis of the most complex polyketides. They are composed of one or more functional and structural modules, each capable of carrying out one step of precursor elongation during the formation of an extended polyketide product. In this thesis, I address two fundamental and open questions in the biosynthesis of polyketides: First, what is the unique architecture underlying the assembly line logic of multimodular PKS assembly lines; and second, how is atomic accuracy achieved in cyclization and aromatic ring formation in the final step of PKS action. The first aim is addressed in chapter two, which provides for the first time detailed structural insights into the organization of type I PKS multimodules. This is achieved by cryo-electron microscopic analysis of filamentous and non-filamentous forms of K3DAK4, a bimodular trans-acyltransferase (AT) PKS fragment from Brevibacillus brevis. Overall reconstructions are provided at an intermediate resolution of 7 Å, with detailed insights into individual domains at sub-3Å resolution from cryo-electron microscopy and X-ray crystallography. The bimodule core displays a vertical stacking of its two modules along the central dimer axis of all three enzymatic domains involved. Additionally, K3DAK4 oligomerizes into filaments horizontally via small scaffolding domains in a trans-AT PKS-specific manner. In chapter three the second aim is tackled, as I visualize an intermediate of the enigmatic targeted cyclization and aromatic ring formation in the product template domain (PT) of the aflatoxin-producing PksA at 2.7 Å resolution using X-ray crystallography. To this end a substrate-analogue mimicking the transient intermediate after the first of two cyclization steps facilitated by the enzyme is covalently crosslinked to the active site. The positioning of the ligand relative to previously known ligands representing the pre-and post-cyclization states indicate an outward movement of the substrate throughout the process and a substantial effect of progressing cyclization on the meticulous positioning of the intermediates. The work provides detailed insights into core aspects of PKS biology from the atomistic picture of guided product modification to the giant overall assembly line architecture. In chapter four, both of these levels are put into context with current advances in the analysis of modular structure and dynamics of PKSs, such as recent structural models of cis-AT PKS modules and iterative PKSs. Furthermore, it addresses currently open questions, such as the interaction of trans-AT PKS with their cognate trans-acting enzymes. Altogether, the current progress in mechanistic understanding of PKS systems makes systematic and structure-guided efforts to unleash the full potential of PKS bioengineering ever more achievable