Structural basis for RNA-duplex unwinding by the DEAD-box helicase DbpA

DEAD-box RNA helicases are implicated in most aspects of RNA biology, where these enzymes unwind short RNA duplexes in an ATP-dependent manner. During the central step of the unwinding cycle, the two domains of the helicase core form a distinct closed conformation that destabilizes the RNA duplex, which ultimately leads to duplex melting. Despite the importance of this step for the unwinding process no high-resolution structures of this state are available. Here, I used nuclear magnetic resonance spectroscopy and X-ray crystallography to determine structures of the DEAD-box helicase DbpA in the closed conformation, complexed with substrate duplexes and single-stranded unwinding product. These structures reveal that DbpA initiates duplex unwinding by interacting with up to three base-paired nucleotides and a 5′ single-stranded RNA duplex overhang. These high-resolution snapshots, together with biochemical assays, rationalize the destabilization of the RNA duplex and are integrated into a conclusive model of the unwinding process

Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone

Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for "heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA-protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperon

A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein TBsmr

AbstractTBsmr is a secondary active multidrug transporter from Mycobacterium tuberculosis that transports a plethora of compounds including antibiotics and fluorescent dyes. It belongs to the small multidrug resistance (SMR) superfamily and is structurally and functionally related to E. coli EmrE. Of particular importance is the link between protein function, oligomeric state and lipid composition. By freeze fracture EM, we found three different size distributions in three different lipid environments for TBsmr indicating different oligomeric states. The link of these states with protein activity has been probed by fluorescence spectroscopy revealing significant differences. The drug binding site has been probed further by 19F-MAS NMR through chemical labeling of native cysteine residues showing a water accessible environment in agreement with the alternating access model

Observation of conformational changes that underlie the catalytic cycle of Xrn2

Nuclear magnetic resonance (NMR) methods that quantitatively probe motions on molecular and atomic levels have propelled the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. In this work, we studied the structure and dynamics of the essential 100-kDa eukaryotic 5′→3′ exoribonuclease Xrn2. A combination of complementary fluorine and methyl-TROSY NMR spectroscopy reveals that the apo enzyme is highly dynamic around the catalytic center. These observed dynamics are in agreement with a transition of the enzyme from the ground state into a catalytically competent state. We show that the conformational equilibrium in Xrn2 shifts substantially toward the active state in the presence of substrate and magnesium. Finally, our data reveal that the dynamics in Xrn2 correlate with the RNA degradation rate, as a mutation that attenuates motions also affects catalytic activity. In that light, our results stress the importance of studies that go beyond static structural information

Insights into the Structure of Invisible Conformations of Large Methyl Group Labeled Molecular Machines from High Pressure NMR

Most proteins are highly flexible and can adopt conformations that deviate from the energetically most favorable ground state. Structural information on these lowly populated, alternative conformations is often lacking, despite the functional importance of these states. Here, we study the pathway by which the Dcp1:Dcp2 mRNA decapping complex exchanges between an autoinhibited closed and an open conformation. We make use of methyl Carr–Purcell–Meiboom–Gill (CPMG) NMR relaxation dispersion (RD) experiments that report on the population of the sparsely populated open conformation as well as on the exchange rate between the two conformations. To obtain volumetric information on the open conformation as well as on the transition state structure we made use of RD measurements at elevated pressures. We found that the open Dcp1:Dcp2 conformation has a lower molecular volume than the closed conformation and that the transition state is close in volume to the closed state. In the presence of ATP the volume change upon opening of the complex increases and the volume of the transition state lies in-between the volumes of the closed and open state. These findings show that ATP has an effect on the volume changes that are associated with the opening-closing pathway of the complex. Our results highlight the strength of pressure dependent NMR methods to obtain insights into structural features of protein conformations that are not directly observable. As our work makes use of methyl groups as NMR probes we conclude that the applied methodology is also applicable to high molecular weight complexes

The ribosome assembly factor Nep1 responsible for Bowen–Conradi syndrome is a pseudouridine-N1-specific methyltransferase

Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen–Conradi syndrome—a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910–921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Ψ) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Ψ in eukaryotic 18S rRNAs

The Bowen–Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen–Conradi syndrome (BCS) is caused by a specific Nep1D86G mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific 14C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1ts mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Assignment of the Ile, Leu, Val, Met and Ala methyl group resonances of the DEAD-box RNA helicase DbpA from E. coli

ATP-dependent DEAD-box helicases constitute one of the largest families of RNA helicases and are important regulators of most RNA-dependent cellular processes. The functional core of these enzymes consists of two RecA-like domains. Changes in the interdomain orientation of these domains upon ATP and RNA binding result in the unwinding of double-stranded RNA. The DEAD-box helicase DbpA from E. coli is involved in ribosome maturation. It possesses a C-terminal RNA recognition motif (RRM) in addition to the canonical RecA-like domains. The RRM recruits DbpA to nascent ribosomes by binding to hairpin 92 of the 23S rRNA. To follow the conformational changes of Dbpa during the catalytic cycle we initiated solution state NMR studies. We use a divide and conquer approach to obtain an almost complete resonance assignment of the isoleucine, leucine, valine, methionine and alanine methyl group signals of full length DbpA (49 kDa). In addition, we also report the backbone resonance assignments of two fragments of DbpA that were used in the course of the methyl group assignment. These assignments are the first step towards a better understanding of the molecular mechanism behind the ATP-dependent RNA unwinding process catalyzed by DEAD-box helicases