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- PDB-7o0h: Structure of the foamy viral protease-reverse transcriptase dRH i... -

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Basic information

Entry
Database: PDB / ID: 7o0h
TitleStructure of the foamy viral protease-reverse transcriptase dRH in complex with ds DNA.
Components
  • DNA (5'-D(*AP*AP*CP*AP*GP*AP*GP*TP*GP*CP*GP*AP*CP*AP*C)-3')
  • DNA (5'-D(*GP*TP*GP*TP*CP*GP*CP*AP*CP*TP*CP*TP*G)-3')
  • Pr125Pol
KeywordsVIRAL PROTEIN / reverse trancscriptase / complex with dsDNA
Function / homology
Function and homology information


virion component / DNA integration / viral penetration into host nucleus / RNA-directed DNA polymerase activity / host cell / RNA-DNA hybrid ribonuclease activity / DNA recombination / host cell cytoplasm / nucleic acid binding / aspartic-type endopeptidase activity ...virion component / DNA integration / viral penetration into host nucleus / RNA-directed DNA polymerase activity / host cell / 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) / Pro-Pol polyprotein
Similarity search - Component
Biological speciesWhite-tufted-ear marmoset simian foamy virus
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.09 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
Revision 1.2May 1, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)143,2564
Polymers143,2564
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Dimer upon binding dsDNa
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8980 Å2
ΔGint-25 kcal/mol
Surface area53230 Å2
MethodPISA
Unit cell
Length a, b, c (Å)158.516, 107.558, 118.090
Angle α, β, γ (deg.)90.000, 98.720, 90.000
Int Tables number5
Space group name H-MC121
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z

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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: 67342.812 Da / Num. of mol.: 2
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: DNA chain DNA (5'-D(*AP*AP*CP*AP*GP*AP*GP*TP*GP*CP*GP*AP*CP*AP*C)-3')


Mass: 4612.026 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*GP*TP*GP*TP*CP*GP*CP*AP*CP*TP*CP*TP*G)-3')


Mass: 3958.571 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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: 3.47 Å3/Da / Density % sol: 64.58 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: PEG 3350, DL malic acid

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, DESY / Beamline: P11 / Wavelength: 1.0332 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Dec 14, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 3.09→50 Å / Num. obs: 35709 / % possible obs: 99.6 % / Redundancy: 3.8 % / Biso Wilson estimate: 86.18 Å2 / CC1/2: 0.994 / Net I/σ(I): 9.3
Reflection shellResolution: 3.09→3.28 Å / Num. unique obs: 5612 / CC1/2: 0.564

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Processing

Software
NameVersionClassification
PHENIX1.19_4092refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: MFV RT

Resolution: 3.09→48.84 Å / SU ML: 0.47 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 32.47 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2853 1786 5 %
Rwork0.2298 33904 -
obs0.2326 35690 99.38 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 239.79 Å2 / Biso mean: 104.9667 Å2 / Biso min: 36.74 Å2
Refinement stepCycle: final / Resolution: 3.09→48.84 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8341 569 0 0 8910
Num. residues----1117
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 13

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.09-3.180.34361300.34572457258795
3.18-3.270.39851370.341125992736100
3.27-3.380.41321370.335526102747100
3.38-3.50.36251370.299125952732100
3.5-3.640.32581370.276626122749100
3.64-3.80.31151380.268426212759100
3.8-40.31661370.243526012738100
4-4.250.28671380.20626102748100
4.25-4.580.2241380.184526302768100
4.58-5.040.24451380.187826242762100
5.04-5.770.27071380.209726242762100
5.77-7.270.25361400.23326482788100
7.27-48.840.26171410.19672673281499
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)
16.1297-1.4441-1.11926.94481.89492.3495-0.0772-0.8493-0.39860.5321-0.0415-1.0215-1.18591.12210.11661.0336-0.2702-0.32410.80480.2680.9968190.257915.4258123.3826
22.3479-0.9995-0.00983.3786-0.07143.0827-0.13740.18690.52810.1311-0.08910.5695-0.7085-0.2540.15670.70290.001-0.03750.3620.07210.9379166.24747.1044117.7609
32.4992-0.2676-1.05692.76340.06235.2309-0.0887-0.1420.22710.54340.11860.0942-0.5215-0.0441-0.00360.52220.0496-0.04950.27360.04770.7014165.1214-0.7892127.0043
41.71690.05080.15922.1646-1.163.8964-0.0681-0.5361-0.20010.56760.24470.2077-0.0521-0.7569-0.17340.72960.04980.22480.70990.12030.6442150.9021-21.9037148.2763
51.3561-0.4483-0.58432.0625-1.451.6662-0.13810.35370.05670.2901-0.9161-1.16650.09882.08760.98080.77040.1921-0.06611.58620.58091.5206201.5299-5.5814126.2051
60.8792-0.1005-0.771.2055-0.83832.1322-0.1338-0.6484-0.17530.5749-0.1668-0.3610.41280.81010.25440.96870.1742-0.21980.93690.18180.8913189.2627-28.7913154.5393
74.07063.4627-3.90039.4248-1.26744.37640.1986-0.0026-0.71660.29740.24380.95490.83220.1194-0.48160.6221-0.0578-0.15050.6530.05691.0372157.8942-25.9994129.1996
83.9572-1.38380.76455.52580.9142.23750.3251-0.0026-1.20.6019-0.21420.6710.7256-0.2425-0.15630.7887-0.2272-0.03450.65080.01460.973157.1942-25.3433131.6448
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 5 through 75 )A5 - 75
2X-RAY DIFFRACTION2chain 'A' and (resid 76 through 202 )A76 - 202
3X-RAY DIFFRACTION3chain 'A' and (resid 203 through 357 )A203 - 357
4X-RAY DIFFRACTION4chain 'A' and (resid 358 through 571 )A358 - 571
5X-RAY DIFFRACTION5chain 'B' and (resid 3 through 104 )B3 - 104
6X-RAY DIFFRACTION6chain 'B' and (resid 105 through 578 )B105 - 578
7X-RAY DIFFRACTION7chain 'E' and (resid 1 through 15 )E1 - 15
8X-RAY DIFFRACTION8chain 'F' and (resid 1 through 13 )F1 - 13

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