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- PDB-8b9j: Cryo-EM structure of MLE in complex with ADP:AlF4 -

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Basic information

Entry
Database: PDB / ID: 8b9j
TitleCryo-EM structure of MLE in complex with ADP:AlF4
ComponentsDosage compensation regulator
KeywordsRNA BINDING PROTEIN / RNA Helicase / Drosophila dosage compensation
Function / homology
Function and homology information


RIP-mediated NFkB activation via ZBP1 / X chromosome located dosage compensation complex, transcription activating / 3'-5' DNA/RNA helicase activity / DEx/H-box helicases activate type I IFN and inflammatory cytokines production / male courtship behavior, veined wing generated song production / regulation of cytoplasmic translation / regulatory region RNA binding / sex-chromosome dosage compensation / PKR-mediated signaling / MSL complex ...RIP-mediated NFkB activation via ZBP1 / X chromosome located dosage compensation complex, transcription activating / 3'-5' DNA/RNA helicase activity / DEx/H-box helicases activate type I IFN and inflammatory cytokines production / male courtship behavior, veined wing generated song production / regulation of cytoplasmic translation / regulatory region RNA binding / sex-chromosome dosage compensation / PKR-mediated signaling / MSL complex / dosage compensation by hyperactivation of X chromosome / positive regulation of DNA topoisomerase (ATP-hydrolyzing) activity / polytene chromosome / 3'-5' RNA helicase activity / regulation of mRNA processing / axon extension / lncRNA binding / 3'-5' DNA helicase activity / DNA duplex unwinding / nuclear chromosome / positive regulation of heterochromatin formation / X chromosome / DNA helicase activity / helicase activity / determination of adult lifespan / double-stranded RNA binding / chromosome / double-stranded DNA binding / RNA helicase activity / RNA helicase / ribonucleoprotein complex / chromatin binding / chromatin / nucleolus / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / RNA binding / ATP binding / nucleus / cytosol
Similarity search - Function
DHX9, first double-stranded RNA binding domain / DHX9, second double-stranded RNA binding domain / DHX9, DEXH-box helicase domain / : / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase-associated domain / Helicase associated domain (HA2), winged-helix / Helicase associated domain (HA2) Add an annotation ...DHX9, first double-stranded RNA binding domain / DHX9, second double-stranded RNA binding domain / DHX9, DEXH-box helicase domain / : / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase-associated domain / Helicase associated domain (HA2), winged-helix / Helicase associated domain (HA2) Add an annotation / Double-stranded RNA binding motif / Double-stranded RNA binding motif / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / TETRAFLUOROALUMINATE ION / Dosage compensation regulator
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsJagtap, P.K.A. / Hennig, J.
Funding support Germany, 1items
OrganizationGrant numberCountry
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND Germany
CitationJournal: Mol Cell / Year: 2023
Title: Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless.
Authors: Pravin Kumar Ankush Jagtap / Marisa Müller / Anna E Kiss / Andreas W Thomae / Karine Lapouge / Martin Beck / Peter B Becker / Janosch Hennig /
Abstract: RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We ...RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies. We discovered that MLE exists in a dsRNA-bound open conformation and that the auxiliary dsRBD2 domain aligns the substrate RNA with the accessible helicase tunnel. In an ATP-dependent manner, dsRBD2 associates with the helicase module, leading to tunnel closure around ssRNA. Furthermore, our structures provide a rationale for blunt-ended dsRNA unwinding and 3'-5' translocation by MLE. Structure-based MLE mutations confirm the functional relevance of our model for RNA unwinding. Our findings contribute to our understanding of the fundamental mechanics of auxiliary domains in DExH helicase MLE, which serves as a model for its human ortholog and potential therapeutic target, DHX9/RHA.
History
DepositionOct 6, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 18, 2023Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Dosage compensation regulator
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,0204
Polymers130,4651
Non-polymers5543
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1230 Å2
ΔGint-15 kcal/mol
Surface area35860 Å2
MethodPISA

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Components

#1: Protein Dosage compensation regulator / ATP-dependent RNA helicase mle / Protein male-less / Protein maleless / Protein no action potential


Mass: 130465.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: mle, nap, CG11680 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P24785, RNA helicase
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: MLE in complex with ADP:AlF4 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.130000 MDa / Experimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Details: 20 mM Tris pH 7.5, 50 mM NaCl, 1mM DTT, 1mM ADP, 1mM AlF3, 10 mM NaF, 2mM MgCl2, 0.5% glycerol, 0.005% Triton X-100
SpecimenConc.: 0.612 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 51.7 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: SerialEM / Version: 4.0.9 / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 234365 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047008
ELECTRON MICROSCOPYf_angle_d0.7589498
ELECTRON MICROSCOPYf_dihedral_angle_d5.832946
ELECTRON MICROSCOPYf_chiral_restr0.0451092
ELECTRON MICROSCOPYf_plane_restr0.0071219

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