scholarly journals Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Marcel J Tauchert ◽  
Jean-Baptiste Fourmann ◽  
Reinhard Lührmann ◽  
Ralf Ficner
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Bound State ◽  
Rna Helicases ◽  
Large Rearrangements ◽  
Rna Complex ◽  
Structural Insights ◽  
Terminal Domains

The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43•ATP-analog•RNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a β-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases.

scholarly journals Structural basis for DEAH-helicase activation by G-patch proteins

2020 ◽  
Vol 117 (13) ◽  
pp. 7159-7170 ◽  
Author(s):  
Michael K. Studer ◽  
Lazar Ivanović ◽  
Marco E. Weber ◽  
Sabrina Marti ◽  
Stefanie Jonas
Keyword(s):  
Conformational Changes ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Rna Helicases ◽  
Catalytic Core ◽  
Terminal Domains

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA–protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch–mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

scholarly journals Structure and mechanism of the PilF DNA transformation ATPase from Thermus thermophilus

2013 ◽  
Vol 450 (2) ◽  
pp. 417-425 ◽  
Author(s):  
Richard F. Collins ◽  
Darin Hassan ◽  
Vijaykumar Karuppiah ◽  
Angela Thistlethwaite ◽  
Jeremy P. Derrick
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Thermus Thermophilus ◽  
Critical Role ◽  
Enzyme Analysis ◽  
Dna Uptake ◽  
Dna And Rna ◽  
Terminal Domains

Many Gram-negative bacteria contain specific systems for uptake of foreign DNA, which play a critical role in the acquisition of antibiotic resistance. The TtPilF (PilF ATPase from Thermus thermophilus) is required for high transformation efficiency, but its mechanism of action is unknown. In the present study, we show that TtPilF is able to bind to both DNA and RNA. The structure of TtPilF was determined by cryoelectron microscopy in the presence and absence of the ATP analogue p[NH]ppA (adenosine 5′-[β,γ-imido]triphosphate), at 10 and 12 Å (1 Å=0.1 nm) resolutions respectively. It consists of two distinct N- and C-terminal regions, separated by a short stem-like structure. Binding of p[NH]ppA induces structural changes in the C-terminal domains, which are transmitted via the stem to the N-terminal domains. Molecular models were generated for the apoenzyme and p[NH]ppA-bound states in the C-terminal regions by docking of a model based on a crystal structure from a closely related enzyme. Analysis of DNA binding by electron microscopy, using gold labelling, localized the binding site to the N-terminal domains. The results suggest a model in which DNA uptake by TtPilF is powered by ATP hydrolysis, causing conformational changes in the C-terminal domains, which are transmitted via the stem to take up DNA into the cell.

scholarly journals Biophysical and Dynamic Characterization of Fine-Tuned Binding of the Human Respiratory Syncytial Virus M2-1 Core Domain to Long RNAs

2020 ◽  
Vol 94 (23) ◽  
Author(s):  
Icaro P. Caruso ◽  
Giovana C. Guimarães ◽  
Vitor B. Machado ◽  
Marcelo A. Fossey ◽  
Dieter Willbold ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Conformational Changes ◽  
Rna Binding ◽  
Contact Region ◽  
Human Respiratory Syncytial Virus ◽  
Dynamic Characterization ◽  
Core Domain ◽  
Syncytial Virus ◽  
Rna Complex

ABSTRACT The human respiratory syncytial virus (hRSV) M2-1 protein functions as a processivity and antitermination factor of the viral polymerase complex. Here, the first evidence that the hRSV M2-1 core domain (cdM2-1) alone has an unfolding activity for long RNAs is presented and the biophysical and dynamic characterization of the cdM2-1/RNA complex is provided. The main contact region of cdM2-1 with RNA was the α1-α2-α5-α6 helix bundle, which suffered local conformational changes and promoted the RNA unfolding activity. This activity may be triggered by base-pairing recognition. RNA molecules wrap around the whole cdM2-1, protruding their termini over the domain. The α2-α3 and α3-α4 loops of cdM2-1 were marked by an increase in picosecond internal motions upon RNA binding, even though they are not directly involved in the interaction. The results revealed that the cdM2-1/RNA complex originates from a fine-tuned binding, contributing to the unraveling interaction aspects necessary for M2-1 activity. IMPORTANCE The main outcome is the molecular description of the fine-tuned binding of the cdM2-1/RNA complex and the provision of evidence that the domain alone has unfolding activity for long RNAs. This binding mode is essential in the understanding of the function in the full-length protein. Human respiratory syncytial virus (hRSV), an orthopneumovirus, stands out for the unique role of its M2-1 protein as a transcriptional antitermination factor able to increase RNA polymerase processivity.

Structural insights into conformational switching in the copper metalloregulator CsoR fromStreptomyces lividans

2015 ◽  
Vol 71 (9) ◽  
pp. 1872-1878 ◽  
Author(s):  
Tatiana V. Porto ◽  
Michael A. Hough ◽  
Jonathan A. R. Worrall
Keyword(s):  
Dna Binding ◽  
Bound States ◽  
Transcriptional Response ◽  
Bound State ◽  
Copper Stress ◽  
Side Chain ◽  
Conformational Switching ◽  
X Ray ◽  
Operator Dna ◽  
Structural Insights

Copper-sensitive operon repressors (CsoRs) act to sense cuprous ions and bind them with a high affinity under copper stress in many bacteria. The binding of copper(I) leads to a conformational change in their homotetramer structure, causing disassembly of the operator DNA–CsoR complex and evoking a transcriptional response. Atomic-level structural insight into the conformational switching mechanism between the apo and metal-bound states is lacking. Here, a new X-ray crystal structure of the CsoR fromStreptomyces lividansis reported and compared with a previously reportedS. lividansCsoR X-ray structure crystallized under different conditions. Based on evidence from this new X-ray structure, it is revealed that the conformational switching between states centres on a concertina effect at the C-terminal end of each α2 helix in the homotetramer. This drives the Cys104 side chain, a copper(I)-ligating residue, into a position enabling copper(I) coordination and as a result disrupts the α2-helix geometry, leading to a compacting and twisting of the homotetramer structure. Strikingly, the conformational switching induces a redistribution of electrostatic surface potential on the tetrameric DNA-binding face, which in the copper(I)-bound state would no longer favour interaction with the mode of operator DNA binding.

scholarly journals Molecular insights into the human ABCB6 transporter

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Guangyuan Song ◽  
Sensen Zhang ◽  
Mengqi Tian ◽  
Laixing Zhang ◽  
Runyu Guo ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Atp Hydrolysis ◽  
Toxic Metal ◽  
Bound State ◽  
Metal Resistance ◽  
Blood Group System ◽  
Cryo Electron Microscopy ◽  
Binding Pockets ◽  
Energy Dependent

AbstractABCB6 plays a crucial role in energy-dependent porphyrin transport, drug resistance, toxic metal resistance, porphyrin biosynthesis, protection against stress, and encoding a blood group system Langereis antigen. However, the mechanism underlying porphyrin transport is still unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structures of nanodisc-reconstituted human ABCB6 trapped in an apo-state and an ATP-bound state at resolutions of 3.6 and 3.5 Å, respectively. Our structures reveal a unique loop in the transmembrane domain (TMD) of ABCB6, which divides the TMD into two cavities. It restrains the access of substrates in the inward-facing state and is removed by ATP-driven conformational change. No ligand cavities were observed in the nucleotide-bound state, indicating a state following substrate release but prior to ATP hydrolysis. Structural analyses and functional characterizations suggest an “ATP-switch” model and further reveal the conformational changes of the substrate-binding pockets triggered by the ATP-driven regulation.

scholarly journals EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation

2017 ◽  
Vol 114 (22) ◽  
pp. E4360-E4369 ◽  
Author(s):  
Maria Hoernke ◽  
Jagan Mohan ◽  
Elin Larsson ◽  
Jeanette Blomberg ◽  
Dana Kahra ◽  
...  
Keyword(s):  
Cell Surface ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Membrane Binding ◽  
Bound State ◽  
Infrared Reflection Absorption Spectroscopy ◽  
Open Conformation ◽  
Infrared Reflection ◽  
Eh Domain ◽  
Membrane Bound

The EH-domain–containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection–absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain–mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.

scholarly journals Cryo-EM structures of lipopolysaccharide transporter LptB2FGC in lipopolysaccharide or AMP-PNP-bound states reveal its transport mechanism

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Xiaodi Tang ◽  
Shenghai Chang ◽  
Qinghua Luo ◽  
Zhengyu Zhang ◽  
Wen Qiao ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Transport Mechanism ◽  
Bound States ◽  
Bound State ◽  
Gram Negative Bacteria ◽  
Transmembrane Helices ◽  
Gram Negative ◽  
Functional Assays ◽  
Shed Light ◽  
Bridge Functional

Abstract Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through a bridge formed by seven membrane proteins (LptBFGCADE). The IM component LptB2FG powers the process through a yet unclarified mechanism. Here we report three high-resolution cryo-EM structures of LptB2FG alone and complexed with LptC (LptB2FGC), trapped in either the LPS- or AMP-PNP-bound state. The structures reveal conformational changes between these states and substrate binding with or without LptC. We identify two functional transmembrane arginine-containing loops interacting with the bound AMP-PNP and elucidate allosteric communications between the domains. AMP-PNP binding induces an inward rotation and shift of the transmembrane helices of LptFG and LptC to tighten the cavity, with the closure of two lateral gates, to eventually expel LPS into the bridge. Functional assays reveal the functionality of the LptF and LptG periplasmic domains. Our findings shed light on the LPS transport mechanism.

scholarly journals Structure and nucleotide-induced conformational dynamics of the Chlorobium tepidum Roco protein

2019 ◽  
Vol 476 (1) ◽  
pp. 51-66 ◽  
Author(s):  
Egon Deyaert ◽  
Margaux Leemans ◽  
Ranjan Kumar Singh ◽  
Rodrigo Gallardo ◽  
Jan Steyaert ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Conformational Dynamics ◽  
Bound State ◽  
Chlorobium Tepidum ◽  
Leucine Rich Repeat ◽  
Hydrogen Deuterium ◽  
Gtpase Cycle ◽  
Hdx Ms ◽  
First Time

Abstract The LRR (leucine-rich repeat)–Roc (Ras of complex proteins)–COR (C-terminal of Roc) domains are central to the action of nearly all Roco proteins, including the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat kinase 2). We previously demonstrated that the Roco protein from Chlorobium tepidum (CtRoco) undergoes a dimer–monomer cycle during the GTPase reaction, with the protein being mainly dimeric in the nucleotide-free and GDP (guanosine-5′-diphosphate)-bound states and monomeric in the GTP (guanosine-5′-triphosphate)-bound state. Here, we report a crystal structure of CtRoco in the nucleotide-free state showing for the first time the arrangement of the LRR–Roc–COR. This structure reveals a compact dimeric arrangement and shows an unanticipated intimate interaction between the Roc GTPase domains in the dimer interface, involving residues from the P-loop, the switch II loop, the G4 region and a loop which we named the ‘Roc dimerization loop’. Hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) is subsequently used to highlight structural alterations induced by individual steps along the GTPase cycle. The structure and HDX-MS data propose a pathway linking nucleotide binding to monomerization and relaying the conformational changes via the Roc switch II to the LRR and COR domains. Together, this work provides important new insights in the regulation of the Roco proteins.

scholarly journals Biophysical and dynamic characterization of a fine-tuned binding of the human Respiratory Syncytial Virus M2-1 core domain to long RNAs

2020 ◽  
Author(s):  
Icaro P. Caruso ◽  
Giovana C. Guimarães ◽  
Vitor B. Machado ◽  
Marcelo A. Fossey ◽  
Dieter Willbold ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Conformational Changes ◽  
Rna Binding ◽  
Contact Region ◽  
Human Respiratory Syncytial Virus ◽  
Dynamic Characterization ◽  
Core Domain ◽  
Syncytial Virus ◽  
Rna Complex

ABSTRACTThe human Respiratory Syncytial Virus (hRSV) M2-1 protein functions as a processivity and antitermination factor of the viral polymerase complex. Here it is presented the first evidence that hRSV M2-1 core domain (cdM2-1) alone has an unfolding activity for long RNAs, as well as a biophysical and dynamic characterization of the cdM2-1/RNA complex. The main contact region of cdM2-1 with RNA was the α1–α2–α5–α6 helix bundle, which suffered local conformational changes and promoted the RNA unfolding activity. This activity may be triggered by base-pairing recognition. RNA molecules wrap around the whole cdM2-1, protruding their terminals over the domain. The α2–α3 and α3–α4 loops of cdM2-1 were marked by an increase in picosecond internal motions upon RNA binding even though they are not directly involved in the interaction. The results revealed that the cdM2-1/RNA complex originates from a fine-tuned binding, contributing to unraveling interaction aspects necessary to M2-1 activity.IMPORTANCEThe main outcome is the molecular description of a fine-tuned binding of the cdM2-1/RNA complex and the evidence that the domain alone has an unfolding activity for long RNAs. This binding mode is essential in the understanding of the function in the full-length protein. Orthopneumovirus, as the human Respiratory Syncytial Virus (hRSV), stands out for the unique role of M2-1 as a transcriptional antitermination factor able to increase the RNA polymerase processivity.

scholarly journals Structural Basis for Katanin Self-Assembly

2017 ◽  
Author(s):  
Stanley Nithiananatham ◽  
Francis J. McNally ◽  
Jawdat Al-Bassam
Keyword(s):  
Conformational Changes ◽  
Self Assembly ◽  
Bound States ◽  
Atp Hydrolysis ◽  
Regulatory Subunit ◽  
Structural Basis ◽  
Inhibition Mechanism ◽  
Atpase Subunit ◽  
Aaa Atpase ◽  
Microtubule Severing

SUMMARYThe reorganization of microtubules in mitosis, meiosis and development requires the microtubule-severing activity of katanin. Katanin is composed of a AAA ATPase subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin’s conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that wild-type katanin only forms heterodimers and heterotetramers. Heterododecamers were only observed for an ATP hydrolysis deficient mutant in the presence of ATP, suggesting an auto-inhibition mechanism that prevents oligomerization. X-ray structures of katanin’s AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states reveal conformational changes in AAA subdomains and N and C-terminal expansion segments that explain this auto-inhibition of assembly. These data lead to a model in which self-inhibited heterodimers bind to a microtubule, then transition into an assembly-competent conformation upon ATP binding. Microtubule-bound heterododecamers then promote tubulin extraction from the microtubule prior to oligomer dissociation.

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