Wieden, Hans-Joachim

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The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome
(Cold Springs Harbor Laboratory Press, 2003) Gu, Shan-Qing; Peske, Frank; Wieden, Hans-Joachim; Rodnina, Marina V.; Wintermeyer, Wolfgang
The signal recognition particle (SRP) from Escherichia coli, composed of Ffh protein and 4.5S RNA, mediates membrane targeting of translating ribosomes displaying a signal or signal-anchor sequence. SRP binds at the peptide exit of the large ribosomal subunit. Structural details of the interaction are not known. Here, the position of Ffh or SRP on the ribosome was probed by using site-specific UV-induced crosslinking by p-azidophenacyl bromide (AzP) attached to a number of cysteine residues engineered into surface positions of Ffh. Efficient crosslinking to vacant ribosomes took place from two positions (AzP17 and AzP25) in the N domain of Ffh, both with Ffh and SRP. Both AzP17 and AzP25 were predominantly crosslinked to ribosomal protein L23 that is located at the peptide exit of the 50S subunit.The SRP receptor, FtsY, did not change the crosslink pattern, whereas the presence of a nascent signal peptide on the ribosome resulted in a second crosslink between Ffh(AzP17) and protein L23, indicating that binding to the nascent signal peptide induced a slightly different arrangement of SRP on the ribosome. These results indicate a model of the topographical arrangement of SRP at the peptide exit of the 50S ribosomal subunit.
De novo cytosine methylation in the differentiating macronucleus of the stichotrichous ciliate Stylonychia lemnae
(Oxford University Press, 2003) Juranek, Stefan; Wieden, Hans-Joachim; Lipps, Hans J.
Dramatic DNA reorganization and elimination processes occur during macronuclear differentiation in ciliates. In this study we analyzed whether cytosine methylation of specific sequences plays a functional role during DNA rearrangement. Three classes of sequences, macronuclear-destined sequences (MDSs, pCE7), members from a large family of transposon-like elements and micronuclear-specific sequences (pLJ01), differing in their structure and future destiny during nuclear differentiation, were studied in the micronucleus, the developing macronucleus and, when present, in the mature macronucleus. While the MDSs become processed to a 1.1 and 1.3 kb gene-sized macronuclear DNA molecule, the family of transposon-like elements represented by MaA81 becomes removed late in the course of polytene chromosome formation. The micronuclear-specific sequence pLJ01 is eliminated together with bulk micronuclear DNA during degradation of polytene chromosomes. No methylated cytosine could be detected in the vegetative macronucleus and no difference in methylation pattern was observed either between micronucleus and developing macronucleus in MDSs or in a micronuclear-speci®c sequence. However, a significant percentage of the cytosines contained in the transposon-like element becomes methylated de novo in the course of macronuclear differentiation. This is the first demonstration that cytosine methylation in speci®c sequences occurs during macronuclear differentiation and may provide a first step towards understanding epigenetic factors involved in DNA processing
An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
(Oxford University Press, 2014) Friedt, Jenna; Leavens, Fern M. V.; Mercier, Evan; Wieden, Hans-Joachim; Wieden-Kothe, Ute
Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by finetuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.
A conserved P-loop anchor limits the structural dynamics that mediate nucleotide dissociation in EF-Tu
(Nature Research, 2015) Mercier, Evan; Girodat, Dylan; Wieden, Hans-Joachim
The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu-GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C.We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.
Identification of two structural elements important for ribosome-dependent GTPase activity of elongation factor 4 (EF4/LepA)
(Nature Research, 2015) De Laurentiis, Evelina I.; Wieden, Hans-Joachim
The bacterial translational GTPase EF4/LepA is structurally similar to the canonical elongation factor EF-G. While sharing core structural features with other translational GTPases, the function of EF4 remains unknown. Recent structural data locates the unique C-terminal domain (CTD) of EF4 in proximity to the ribosomal peptidyl transferase center (PTC). To investigate the functional role of EF4’s CTD we have constructed three C-terminal truncation variants.These variants are fully functional with respect to binding mant-GTP and mant-GDP as determined by rapid kinetics, as well as their intrinsic multiple turnover GTPase activity. Furthermore, they are able to form stable complexes with the 70S ribosome and 50S/30S ribosomal sub units.However,successive removal of the C-terminus impairs ribosome-dependent multiple turnover GTPase activity of EF4, which for the full-length protein is very similar to EF-G. Our findings suggest that the last 44 C-terminal amino acids of EF4 form a sub-domain within the C-terminal domain that is important for GTP-dependent function on the ribosome. Additionally, we show that efficient nucleotide hydrolysis by EF4 on the ribosome depends on a conserved histidine (His 81), similar to EF-G and EF-Tu.
The C-terminal helix of Pseudomonas aeruginosa elongation factor Ts tunes EFT-Tu dynamics to modulate nucleotide exchange
(American Society for Biochemistry and Molecular Biology, 2015) De Laurentiis, Evelina I.; Mercier, Evan; Wieden, Hans-Joachim
Little is known about the conservation of critical kinetic parameters and the mechanistic strategies of elongation factor (EF) Ts-catalyzed nucleotide exchange in EF-Tu in bacteria and particularly in clinically relevant pathogens. EF-Tu from the clinically relevant pathogen Pseudomonas aeruginosa shares over 84% sequence identity with the corresponding elongation factor from Escherichia coli. Interestingly, the functionally closely linked EF-Ts only shares 55% sequence identity. To identify any differences in the nucleotide binding properties, as well as in the EF-Ts-mediated nucleotide exchange reaction, we performed a comparative rapid kinetics and mutagenesis analysis of the nucleotide exchange mechanism for both the E. coli and P. aeruginosa systems, identifying helix 13 of EF-Ts as a previously unnoticed regulatory element in the nucleotide exchange mechanism with species-specific elements. Our findings support the base side-first entry of the nucleotide into the binding pocket of the EF-Tu·EF-Ts binary complex, followed by displacement of helix 13 and rapid binding of the phosphate side of the nucleotide, ultimately leading to the release of EF-Ts.
Contribution of two conserved histidines to the dual activity of archael RNA guide-dependent and -independent pseudouridine synthase Cbf5
(Cold Spring Harbor Laboratory Press, 2015) Tillault, Anne-Sophie; Fourmann, Jean-Baptiste; Loegler, Christine; Wieden, Hans-Joachim; Wieden-Kothe, Ute; Charpentier, Bruno
In all organisms, several distinct stand-alone pseudouridine synthase (PUS) family enzymes are expressed to isomerizeuridine into pseudouridine (Ψ) by specific recognition of RNAs. In addition, Ψs are generated in Archaea and Eukaryotes by PUS enzymes which are organized as ribonucleoprotein particles (RNP)—the box H/ACA s/snoRNPs. For this modification system, a unique TruB-like catalytic PUS subunit is associated with various RNA guides which specifically target and secure substrate RNAs by base-pairing. The archaeal Cbf5 PUS displays the special feature of exhibiting both RNA guide-dependent and -independent activities. Structures of substrate-bound TruB and H/ACA sRNP revealed the importance of histidines in positioning the target uridine in the active site. To analyze the respective role of H60 and H77, we have generated variants carrying alanine substitutions at these positions. The impact of the mutations was analyzed for unguided modifications U55 in tRNA and U2603 in 23S rRNA, and for activity of the box H/ACA Pab91 sRNP enzyme. H77 (H43 in TruB), but not H60, appeared to be crucial for the RNA guide-independent activity. In contrast to earlier suggestions, H60 was found to be noncritical for the activity of the H/ACA sRNP, but contributes together with H77 to the full activity of H/ACA sRNPs. The data suggest that a similar catalytic process was conserved in the two divergent pseudouridylation systems.
Reproducibility of fluorescent expression from engineering biological constructs in E. coli
(Public Library of Science, 2016) Beal, Jacob; Haddock-Angelli, Traci; Gershater, Markus; De Mora, Kim; Lizarazo, Meagan; Hollenhorst, Jim; Rettberg, Randy; iGEM Interlab Study Contributors
We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E.coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.
The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome
(Oxford University Press, 2016) Coatham, Mackenzie L.; Brandon, Harland E.; Fischer, Jeffrey J.; Schummer, Tobias; Wieden, Hans-Joachim
Using a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX.
Using rapid kinetics and molecular dynamics simulations to study biomolecular information processing and design
(Biomath Forum, 2016) De Laurentiis, Evelina I.; Girodat, Dylan J.; Mercier, Evan; Wieden, Hans-Joachim
Translation, the ribosome dependent synthesis of proteins, is the last step of gene expression. It is targeted by a large number of antibiotics that modulate, in one way or the other, the dynamic properties of the involved biomolecules. Protein synthesis is a highly conserved multi-step process facilitated by a large ribonucleoprotein complex (the ribosome) acting as a biomolecular assembler, converting genetic information provided as RNA transcripts (mRNA) into proteins. This process occurs with high speed and incredible accuracy within any living system (1). As such, accuracy is the key to maintaining the integrity of the genetic information to be reflected in the resulting proteins. The underlying design principles that enable the translation machinery to achieve these performance characteristics is of great interest for a large number of fields and applications, naming antibiotics design and the rational engineering of biomolecular machines just as two examples.
Characterization of fluorescein arsenical hairpin (FIAsH) as a probe for single-molecule fluorescence spectroscopy
(Nature Research, 2017) Fernandes, Dennis D.; Bamrah, Jasbir; Kailasam, Senthilkumar; Gomes, Gregory-Neal W.; Li, Yuchong; Wieden, Hans-Joachim; Gradinaru, Claudiu C.
In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.
Tetracycline does not directly inhibit the function of bacterial elongation factor Tu
(Public Library of Science, 2017) Gzyl, Katherine E.; Wieden, Hans-Joachim
Understanding the molecular mechanism of antibiotics that are currently in use is important for the development of new antimicrobials. The tetracyclines, discovered in the 1940s, are a well-established class of antibiotics that still have a role in treating microbial infections in humans. It is generally accepted that the main target of their action is the ribosome. The esti- mated affinity for tetracycline binding to the ribosome is relatively low compared to the actual potency of the drug in vivo . Therefore, additional inhibitory effects of tetracycline on the translation machinery have been discussed. Structural evidence suggests that tetracycline inhibits the function of the essential bacterial GTPase Elongation Factor (EF)-Tu through interaction with the bound nucleotide. Based on this, tetracycline has been predicted to impede the nucleotide-binding properties of EF-Tu. However, detailed kinetic studies addressing the effect of tetracycline on nucleotide binding have been prevented by the fluorescence properties of the antibiotic. Here, we report a fluorescence-bas ed kinetic assay that minimizes the effect of tetracycline autofluorescence, enabling the detailed kinetic analysis of the nucleotide-bin ding properties of Escherichia coli EF-Tu. Further- more, using physiologica lly relevant conditions, we demonstrate that tetracycline does not affect EF-Tu’s intrinsic or ribosome-stimulated GTPase activity, nor the stability of the EF- Tu•GTP•Phe-tRNA Phe complex. We therefore provide clear evidence that tetracycline does not directly impede the function of EF-Tu.
The C-terminal helix of ribosomal P stalk recognizes a hydropobic groove of elongation factor 2 in a novel fashion
(Oxford University Press, 2018) Tanzawa, Takehito; Kato, Koji; Girodat, Dylan; Ose, Toyoyuki; Kumakura, Yuki; Weiden, Hans-Joachim; Uchiumi, Toshio; Tanaka, Isao; Yao, Min
Archaea and eukaryotes have ribosomal P stalks composed of anchor protein P0 and aP1 homodimers (archaea) or P1•P2 heterodimers (eukaryotes). These P stalks recruit translational GTPases to the GTPase-associated center in ribosomes to provide energy during translation. The C-terminus of the P stalk is known to selectively recognize GTPases. Here we investigated the interaction between the P stalk and elongation factor 2 by determining the structures of P y ro c o c c u s h o r i ko s h i i EF-2 ( Pho EF2) in the Apo-form, GDP-form, GMPPCP-form (GTPform), and GMPPCP-form bound with 11 C-terminal residues of P1 (P1C11). Helical structured P1C11 binds to a hydrophobic groove between domain G and subdomain G of Pho EF-2, where is completely differentfromthatofaEF-1 in terms of both position and sequence, implying that such interaction characteristic may be requested by how GTPases perform their functions on the ribosome. Combining Pho EF2 P1-binding assays with a structural comparison of current Pho EF-2 structures and molecular dynamics model of a P1C11-bound GDP form, the conformational changes of the P1C11-binding groove in each form suggest that in response to the translation process, the groove has three states: closed, open, and release for recruiting and releasing GTPases
Cellular roles of the human Obg-like ATPase 1 (hOLA1) and its YchF homologs
(Canadian Science Publishing, 2020) Balasingam, Nirujah; Brandon, Harland E.; Ross, Joseph A.; Wieden, Hans-Joachim; Thakor, Nehal
P-loop NTPases comprise one of the major superfamilies of nucleotide binding proteins, which mediate a variety of cellular processes, such as mRNA translation, signal transduction, cell motility, and growth regulation. In this review, we discuss the structure and function of two members of the ancient Obg-related family of P-loop GTPases: human Obg-like ATPase 1 (hOLA1), and its bacterial/plant homolog, YchF. After a brief discussion of nucleotide binding proteins in general and the classification of the Obg-related family in particular, we discuss the sequence and structural features of YchF and hOLA1. We then explore the various functional roles of hOLA1 in mammalian cells during stress response and cancer progression, and of YchF in bacterial cells. Finally, we directly compare and contrast the structure and function of hOLA1 with YchF before summarizing the future perspectives of hOLA1 research. This review is timely, given the variety of recent studies aimed at understanding the roles of hOLA1 and YchF in such critical processes as cellular-stress response, oncogenesis, and protein synthesis.
Human DDX17 unwinds Rift Valley fever virus non-coding RNAs
(MDPI, 2020) Nelson, Corey R.; Mrozowich, Tyler; Park, Sean M.; D'Souza, Simmone; Henrickson, Amy; Vigar, Justin R. J.; Wieden, Hans-Joachim; Owens, Raymond J.; Demeler, Borries; Patel, Trushar R.
Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5′ non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17135–555) and RVFV non-coding RNAs, IGR and 5’ NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17135–555, IGR, and 5’ NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5’ NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17135–555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17135–555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17135–555 interacting with and unwinding RVFV non-coding regions

Wieden, Hans-Joachim

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