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- PDB-7o0g: Structure of the foamy viral protease-reverse transcriptase in co... -

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Basic information

Entry
Database: PDB / ID: 7o0g
TitleStructure of the foamy viral protease-reverse transcriptase in complex with RNA/DNA hybrid.
Components
  • DNA (5'-D(*CP*CP*TP*CP*TP*CP*CP*TP*GP*GP*AP*CP*AP*AP*G)-3')
  • Pr125Pol
  • RNA (5'-R(*UP*UP*CP*UP*UP*GP*UP*CP*CP*AP*GP*GP*AP*GP*AP*GP*G)-3')
KeywordsVIRAL PROTEIN / reverse trancscriptase / complex with RNA-DNA
Function / homology
Function and homology information


virion component / DNA integration / viral penetration into host nucleus / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / DNA recombination / host cell cytoplasm / nucleic acid binding / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity ...virion component / DNA integration / viral penetration into host nucleus / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / DNA recombination / host cell cytoplasm / nucleic acid binding / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / proteolysis / metal ion binding / cytoplasm
Similarity search - Function
Spumavirus aspartic protease A9 / Retroviral integrase, C-terminal SH3 domain / Spumavirus aspartic protease (A9) / Retroviral integrase C-terminal SH3 domain / Foamy virus protease (FV PR) domain profile. / Integrase zinc-binding domain / Integrase zinc binding domain / Reverse transcriptase/retrotransposon-derived protein, RNase H-like domain / RNase H-like domain found in reverse transcriptase / RNase H ...Spumavirus aspartic protease A9 / Retroviral integrase, C-terminal SH3 domain / Spumavirus aspartic protease (A9) / Retroviral integrase C-terminal SH3 domain / Foamy virus protease (FV PR) domain profile. / Integrase zinc-binding domain / Integrase zinc binding domain / Reverse transcriptase/retrotransposon-derived protein, RNase H-like domain / RNase H-like domain found in reverse transcriptase / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Ribonuclease H domain / RNase H type-1 domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Pro-Pol polyprotein
Similarity search - Component
Biological speciesWhite-tufted-ear marmoset simian foamy virus
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 3.1 Å
AuthorsNowak, E. / Nowacka, M. / Nowotny, M.
Funding support Poland, 1items
OrganizationGrant numberCountry
Polish National Science CentreUMO-2016/21/B/NZ1/02757 Poland
CitationJournal: J Virol / Year: 2021
Title: Structures of Substrate Complexes of Foamy Viral Protease-Reverse Transcriptase.
Authors: Marzena Nowacka / Elżbieta Nowak / Mariusz Czarnocki-Cieciura / Justyna Jackiewicz / Krzysztof Skowronek / Roman H Szczepanowski / Birgitta M Wöhrl / Marcin Nowotny /
Abstract: Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of ...Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: and . The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA.
History
DepositionMar 26, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 30, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 19, 2022Group: Database references / Category: citation / citation_author / database_2
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 / _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pr125Pol
E: RNA (5'-R(*UP*UP*CP*UP*UP*GP*UP*CP*CP*AP*GP*GP*AP*GP*AP*GP*G)-3')
F: DNA (5'-D(*CP*CP*TP*CP*TP*CP*CP*TP*GP*GP*AP*CP*AP*AP*G)-3')


Theoretical massNumber of molelcules
Total (without water)95,8013
Polymers95,8013
Non-polymers00
Water181
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Monomer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4470 Å2
ΔGint-27 kcal/mol
Surface area40380 Å2
MethodPISA
Unit cell
Length a, b, c (Å)105.028, 105.028, 253.525
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

#1: Protein Pr125Pol / Pro-Pol polyprotein / Protease/Reverse transcriptase / Protease/Reverse transcriptase/ribonuclease ...Pro-Pol polyprotein / Protease/Reverse transcriptase / Protease/Reverse transcriptase/ribonuclease H / Ribonuclease H / p42In / p65Pro-RT / p87Pro-RT-RNaseH


Mass: 85465.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: foamy virus
Source: (gene. exp.) White-tufted-ear marmoset simian foamy virus
Gene: pol
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: D5JWV1, RNA-directed DNA polymerase, DNA-directed DNA polymerase, ribonuclease H
#2: RNA chain RNA (5'-R(*UP*UP*CP*UP*UP*GP*UP*CP*CP*AP*GP*GP*AP*GP*AP*GP*G)-3')


Mass: 5805.476 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Electron density was not observed for the last G / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*CP*CP*TP*CP*TP*CP*CP*TP*GP*GP*AP*CP*AP*AP*G)-3')


Mass: 4529.950 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.65 Å3/Da / Density % sol: 66.29 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.5 / Details: PEG 20000, PEG MME550, MES/imidazole

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.97931 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Aug 26, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97931 Å / Relative weight: 1
ReflectionResolution: 3.1→50 Å / Num. obs: 26673 / % possible obs: 99.8 % / Redundancy: 13.1 % / Biso Wilson estimate: 119.84 Å2 / CC1/2: 0.998 / Net I/σ(I): 21.1
Reflection shellResolution: 3.1→3.28 Å / Redundancy: 13.5 % / Num. unique obs: 4179 / CC1/2: 0.821 / % possible all: 99.3

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Processing

Software
NameVersionClassification
PHENIX1.19_4092refinement
PHENIX1.19_4092refinement
XDSdata reduction
XSCALEdata scaling
CRANK2phasing
RefinementMethod to determine structure: SAD / Resolution: 3.1→48.52 Å / SU ML: 0.5481 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 33.4813
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.273 1329 4.99 %
Rwork0.2474 25311 -
obs0.2487 26640 99.71 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 126.78 Å2
Refinement stepCycle: LAST / Resolution: 3.1→48.52 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5569 661 0 1 6231
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00296492
X-RAY DIFFRACTIONf_angle_d0.57899027
X-RAY DIFFRACTIONf_chiral_restr0.04141064
X-RAY DIFFRACTIONf_plane_restr0.00531028
X-RAY DIFFRACTIONf_dihedral_angle_d12.79661140
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.220.47571400.40372698X-RAY DIFFRACTION98.68
3.22-3.370.34611450.34492765X-RAY DIFFRACTION99.9
3.37-3.540.35381450.33092756X-RAY DIFFRACTION99.59
3.54-3.770.32831470.32032783X-RAY DIFFRACTION99.8
3.77-4.060.3261460.28092777X-RAY DIFFRACTION99.9
4.06-4.460.27361470.24652790X-RAY DIFFRACTION99.86
4.46-5.110.30031490.23382833X-RAY DIFFRACTION100
5.11-6.430.27521500.25872872X-RAY DIFFRACTION99.97
6.43-48.520.21461600.19723037X-RAY DIFFRACTION99.69
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.644353645220.8067011951080.9005989180260.8403794074970.7594244968665.45502276370.4512005315570.152343689677-0.3570138216530.0219331501969-0.3095473526780.22472269366-0.0162673053303-0.278556009905-0.1640802865891.07750804611-0.09903189704310.0253157271680.756338076194-0.05771481719391.0430617625216.5155930679-27.1741561793-25.0587697609
25.947601873951.92720512094-2.025562097441.84033089504-1.870088555213.944439445330.198344251924-0.291911199221-0.2270844551040.285774446823-0.2229377977160.06717584513570.3287446453050.1562669467630.008792760973680.978708495262-0.0876877671790.007150163715490.556536794877-0.03399464074670.67886266671831.8631421102-19.2560024026-11.7868489386
36.99822205307-3.68904027907-3.273358408546.169569167944.099091824767.72705567731-0.155235754425-0.839059747795-0.3840174905140.8224303278720.387198878723-0.6350839218361.184467161481.10102190795-0.3435116245241.222569979890.161503968065-0.1337742351440.9697664556540.003320375062210.71636156807248.4277612651-12.675204310614.2976241417
42.15968008905-0.2499011308781.803568701390.6616073448550.06843559524563.38319932156-0.07539831977760.189063093446-0.0174444582171-0.0241947123056-0.00371693961098-0.05541507678650.1151828805390.28246095930.02644004551820.8942746771430.08648864058080.08907658365570.831868779602-0.04832903122650.66605215038653.60937484753.8536349708428.7526841496
56.60778190342-6.4976448114-4.445122201029.799953663867.530554065157.15252869265-1.82679778292-0.210394256737-0.3404645344292.326872184010.6961396452070.2177564913950.6110508840891.958618780891.003454957141.738702648350.0740528695568-0.09767626820891.44559747998-0.0337794756390.80208476024642.4588937399-11.062135518-8.03353031925
62.7389296245-1.27747431353-2.57625703524.758255284092.972327327993.19935787814-1.27871680711-0.513939771979-1.26328774474-0.7792161799730.04582603291620.251241690225-1.747538323590.9534075615471.283083156741.169626149480.0123204183761-0.05387587469420.9956565391570.1684316793731.092861785648.2871883736-3.771690911861.98140165415
78.64537325956-5.636662368952.320063039834.70457555406-3.120368123873.125507036481.69606560002-0.0117506417606-1.72038315397-0.615625524628-1.026206160192.68201501386-0.3901569708750.604310305822-0.6110608483081.40780924477-0.171200152733-0.08766771448311.34601389598-0.1381357076491.4627503956660.22263198758.92131230702-6.48141526484
89.720227176876.376711717037.206519750594.516684225094.830652910235.375909957853.10001068789-1.08951203345-2.381608805452.36089050530.422206334151-0.8331655398622.600606238750.203301001041-3.477192216962.16077741091-0.45332301873-0.3771831998671.79865322197-0.005902846219462.5072590130158.52806496221.89129296970.897051662288
91.39386296121-0.061067431885-2.507100132497.61727031978-3.735343129856.815190253290.479910468605-0.6902894238840.1376903655650.435061529315-0.6099377015840.702645832203-0.03289678467590.2799250913380.1109216209950.773188677686-0.119087216427-0.07653490526991.44490927102-0.1111480220550.91790602590950.81711183380.456771871048-2.21628948452
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 4 through 132 )AA4 - 1321 - 128
22chain 'A' and (resid 133 through 370 )AA133 - 370129 - 364
33chain 'A' and (resid 371 through 443 )AA371 - 443365 - 437
44chain 'A' and (resid 444 through 749 )AA444 - 749438 - 733
55chain 'E' and (resid 1 through 5 )EB1 - 5
66chain 'E' and (resid 6 through 10 )EB6 - 10
77chain 'E' and (resid 11 through 15 )EB11 - 15
88chain 'E' and (resid 16 through 17 )EB16 - 17
99chain 'F' and (resid 1 through 15 )FC1 - 15

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