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- PDB-8c8j: Long Interspersed Nuclear Element 1 (LINE-1) reverse transcriptas... -

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Basic information

Entry
Database: PDB / ID: 8c8j
TitleLong Interspersed Nuclear Element 1 (LINE-1) reverse transcriptase ternary complex with hybrid duplex and dTTP
Components
  • DNA (5'-D(P*GP*CP*GP*CP*TP*TP*TP*CP*C)-3')
  • RNA (5'-R(P*UP*UP*AP*GP*GP*AP*AP*AP*GP*CP*GP*C)-3')
  • RNA-directed DNA polymerase
KeywordsREPLICATION / REVERSE TRANSCRIPTASE / RT / DTTP / HYRBID DUPLEX / TEMPLATE PRIMER
Function / homology
Function and homology information


catalytic activity / RNA-directed DNA polymerase
Similarity search - Function
Endonuclease/exonuclease/phosphatase / Endonuclease/Exonuclease/phosphatase family / Endonuclease/exonuclease/phosphatase superfamily / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
CITRIC ACID / 1,4-DIETHYLENE DIOXIDE / THYMIDINE-5'-TRIPHOSPHATE / DNA / RNA / RNA (> 10) / RNA-directed DNA polymerase
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsNichols, C.E. / Walpole, T.B. / Baldwin, E.
Funding support United States, 1items
OrganizationGrant numberCountry
ROME Therapeutics, Inc. United States
CitationJournal: Nature / Year: 2024
Title: Structures, functions and adaptations of the human LINE-1 ORF2 protein.
Authors: Eric T Baldwin / Trevor van Eeuwen / David Hoyos / Arthur Zalevsky / Egor P Tchesnokov / Roberto Sánchez / Bryant D Miller / Luciano H Di Stefano / Francesc Xavier Ruiz / Matthew Hancock / ...Authors: Eric T Baldwin / Trevor van Eeuwen / David Hoyos / Arthur Zalevsky / Egor P Tchesnokov / Roberto Sánchez / Bryant D Miller / Luciano H Di Stefano / Francesc Xavier Ruiz / Matthew Hancock / Esin Işik / Carlos Mendez-Dorantes / Thomas Walpole / Charles Nichols / Paul Wan / Kirsi Riento / Rowan Halls-Kass / Martin Augustin / Alfred Lammens / Anja Jestel / Paula Upla / Kera Xibinaku / Samantha Congreve / Maximiliaan Hennink / Kacper B Rogala / Anna M Schneider / Jennifer E Fairman / Shawn M Christensen / Brian Desrosiers / Gregory S Bisacchi / Oliver L Saunders / Nafeeza Hafeez / Wenyan Miao / Rosana Kapeller / Dennis M Zaller / Andrej Sali / Oliver Weichenrieder / Kathleen H Burns / Matthias Götte / Michael P Rout / Eddy Arnold / Benjamin D Greenbaum / Donna L Romero / John LaCava / Martin S Taylor /
Abstract: The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open ...The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p). ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer, autoimmunity and ageing, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.
History
DepositionJan 20, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 31, 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Feb 14, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-directed DNA polymerase
B: DNA (5'-D(P*GP*CP*GP*CP*TP*TP*TP*CP*C)-3')
C: RNA (5'-R(P*UP*UP*AP*GP*GP*AP*AP*AP*GP*CP*GP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,64326
Polymers104,5793
Non-polymers2,06423
Water3,945219
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, LINE1-RT elution from gel filtration purification step is consistent with monomer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10000 Å2
ΔGint-3 kcal/mol
Surface area31300 Å2
MethodPISA
Unit cell
Length a, b, c (Å)119.349, 84.528, 107.938
Angle α, β, γ (deg.)90, 91.48, 90
Int Tables number5
Space group name H-MC121

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Components

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Protein / DNA chain / RNA chain , 3 types, 3 molecules ABC

#1: Protein RNA-directed DNA polymerase


Mass: 98020.680 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / References: UniProt: G8I2S9, RNA-directed DNA polymerase
#2: DNA chain DNA (5'-D(P*GP*CP*GP*CP*TP*TP*TP*CP*C)-3')


Mass: 2682.760 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: RNA chain RNA (5'-R(P*UP*UP*AP*GP*GP*AP*AP*AP*GP*CP*GP*C)-3')


Mass: 3875.384 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 8 types, 242 molecules

#4: Chemical ChemComp-CIT / CITRIC ACID


Mass: 192.124 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H8O7
#5: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C2H6O2
#7: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE


Mass: 78.133 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#8: Chemical ChemComp-DIO / 1,4-DIETHYLENE DIOXIDE


Mass: 88.105 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H8O2
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 219 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 6

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Sample preparation

Crystal
IDDensity Matthews (Å3/Da)Density % sol (%)
12.652.73
6
5
4
3
2
Crystal grow
Temperature (K)Crystal-IDMethodpHDetails
2931vapor diffusion, sitting drop5.6PEG8000, sodium citrate, 1,4 dioxane, ethylene glycol, dimethyl sulphoxide
2936vapor diffusion5.6PEG8000, sodium citrate, 1,4 dioxane, ethylene glycol, dimethyl sulphoxide
2935vapor diffusion5.6PEG8000, sodium citrate, 1,4 dioxane, ethylene glycol, dimethyl sulphoxide
2934vapor diffusion5.6PEG8000, sodium citrate, 1,4 dioxane, ethylene glycol, dimethyl sulphoxide
2933vapor diffusion5.6PEG8000, sodium citrate, 1,4 dioxane, ethylene glycol, dimethyl sulphoxide
2932vapor diffusion5.6PEG8000, sodium citrate, 1,4 dioxane, ethylene glycol, dimethyl sulphoxide

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Data collection

Diffraction
IDMean temperature (K)Crystal-IDSerial crystal experiment
11001N
61006N
51005N
41004N
31003N
21002N
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONDiamond I0310.9762
SYNCHROTRONDiamond I0360.9762
SYNCHROTRONDiamond I0350.9762
SYNCHROTRONDiamond I0340.9762
SYNCHROTRONDiamond I0330.9762
SYNCHROTRONDiamond I0320.9762
Detector
TypeIDDetectorDate
DECTRIS EIGER2 XE 16M1PIXELAug 5, 2022
DECTRIS EIGER2 XE 16M6PIXELAug 6, 2022
DECTRIS EIGER2 XE 16M5PIXELAug 6, 2022
DECTRIS EIGER2 XE 16M4PIXELAug 6, 2022
DECTRIS EIGER2 XE 16M3PIXELAug 6, 2022
DECTRIS EIGER2 XE 16M2PIXELAug 5, 2022
Radiation
IDProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1SINGLE WAVELENGTHMx-ray1
6SINGLE WAVELENGTHMx-ray2
5SINGLE WAVELENGTHMx-ray3
4SINGLE WAVELENGTHMx-ray4
3SINGLE WAVELENGTHMx-ray5
2SINGLE WAVELENGTHMx-ray6
Radiation wavelength
IDWavelength (Å)Relative weight
10.97621
21
31
41
51
61
ReflectionResolution: 2.1→59.66 Å / Num. all: 62734 / Num. obs: 62715 / % possible obs: 100 % / Redundancy: 41.2 % / Biso Wilson estimate: 49.1 Å2 / CC1/2: 1 / Rpim(I) all: 0.018 / Net I/σ(I): 18.7
Reflection shellResolution: 2.1→2.15 Å / Redundancy: 42.1 % / Mean I/σ(I) obs: 1.9 / Num. unique obs: 4676 / CC1/2: 0.892 / Rpim(I) all: 0.427 / % possible all: 100

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Processing

Software
NameVersionClassification
BUSTER2.11.8 (8-JUN-2022)refinement
DIALSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.1→59.66 Å / Cor.coef. Fo:Fc: 0.931 / Cor.coef. Fo:Fc free: 0.925 / SU R Cruickshank DPI: 0.182 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.178 / SU Rfree Blow DPI: 0.157 / SU Rfree Cruickshank DPI: 0.16
RfactorNum. reflection% reflectionSelection details
Rfree0.238 3001 -RANDOM
Rwork0.2079 ---
obs0.2093 62715 100 %-
Displacement parametersBiso mean: 61.79 Å2
Baniso -1Baniso -2Baniso -3
1--22.1967 Å20 Å2-9.7979 Å2
2--6.9248 Å20 Å2
3---15.2719 Å2
Refine analyzeLuzzati coordinate error obs: 0.3 Å
Refinement stepCycle: LAST / Resolution: 2.1→59.66 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5573 439 128 219 6359
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0086381HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.898725HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d2171SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes995HARMONIC5
X-RAY DIFFRACTIONt_it6381HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion884SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact5188SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion3.1
X-RAY DIFFRACTIONt_other_torsion17.16
LS refinement shellResolution: 2.1→2.11 Å
RfactorNum. reflection% reflection
Rfree0.2784 62 -
Rwork0.3115 --
obs0.3098 1255 99.92 %

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