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- PDB-7ouh: Structure of the STLV intasome:B56 complex bound to the strand-tr... -

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Basic information

Entry
Database: PDB / ID: 7ouh
TitleStructure of the STLV intasome:B56 complex bound to the strand-transfer inhibitor bictegravir
Components
  • DNA (5'-D(*AP*CP*TP*GP*TP*GP*TP*TP*TP*GP*GP*CP*GP*CP*TP*TP*CP*TP*CP*TP*C)-3')
  • DNA (5'-D(*GP*AP*GP*AP*GP*AP*AP*GP*CP*GP*CP*CP*AP*AP*AP*CP*AP*CP*A)-3')
  • Integrase
  • PC4 and SFRS1-interacting protein,Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform
KeywordsVIRAL PROTEIN / integrase / intasome / HTLV / STLV / integration / strand-transfer inhibitors / INSTI / bictegravir / BIC / drug
Function / homology
Function and homology information


DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / DNA recombination / nucleic acid binding / zinc ion binding
Similarity search - Function
Integrase Zinc binding domain / Integrase DNA binding domain / Integrase, N-terminal zinc-binding domain / Integrase, C-terminal, retroviral / Integrase DNA binding domain profile. / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Ribonuclease H domain ...Integrase Zinc binding domain / Integrase DNA binding domain / Integrase, N-terminal zinc-binding domain / Integrase, C-terminal, retroviral / Integrase DNA binding domain profile. / 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 / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Bictegravir / DNA / DNA (> 10) / Pol protein
Similarity search - Component
Biological speciesSimian T-lymphotropic virus 1
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBarski, M.S. / Ballandras-Colas, A. / Cronin, N.B. / Pye, V.E. / Cherepanov, P. / Maertens, G.N.
Funding support United Kingdom, United States, 4items
OrganizationGrant numberCountry
Wellcome Trust107005/Z/15Z United Kingdom
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)GM082251 United States
The Francis Crick InstituteFC001061 United Kingdom
Wellcome Trust206175/Z/17/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures.
Authors: Michal S Barski / Teresa Vanzo / Xue Zhi Zhao / Steven J Smith / Allison Ballandras-Colas / Nora B Cronin / Valerie E Pye / Stephen H Hughes / Terrence R Burke / Peter Cherepanov / Goedele N Maertens /
Abstract: Between 10 and 20 million people worldwide are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). Despite causing life-threatening pathologies there is no therapeutic regimen for this ...Between 10 and 20 million people worldwide are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). Despite causing life-threatening pathologies there is no therapeutic regimen for this deltaretrovirus. Here, we screened a library of integrase strand transfer inhibitor (INSTI) candidates built around several chemical scaffolds to determine their effectiveness in limiting HTLV-1 infection. Naphthyridines with substituents in position 6 emerged as the most potent compounds against HTLV-1, with XZ450 having highest efficacy in vitro. Using single-particle cryo-electron microscopy we visualised XZ450 as well as the clinical HIV-1 INSTIs raltegravir and bictegravir bound to the active site of the deltaretroviral intasome. The structures reveal subtle differences in the coordination environment of the Mg ion pair involved in the interaction with the INSTIs. Our results elucidate the binding of INSTIs to the HTLV-1 intasome and support their use for pre-exposure prophylaxis and possibly future treatment of HTLV-1 infection.
History
DepositionJun 11, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 18, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-13077
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
D: Integrase
E: Integrase
F: PC4 and SFRS1-interacting protein,Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform
I: DNA (5'-D(*AP*CP*TP*GP*TP*GP*TP*TP*TP*GP*GP*CP*GP*CP*TP*TP*CP*TP*CP*TP*C)-3')
J: DNA (5'-D(*GP*AP*GP*AP*GP*AP*AP*GP*CP*GP*CP*CP*AP*AP*AP*CP*AP*CP*A)-3')
A: Integrase
B: Integrase
C: PC4 and SFRS1-interacting protein,Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform
K: DNA (5'-D(*AP*CP*TP*GP*TP*GP*TP*TP*TP*GP*GP*CP*GP*CP*TP*TP*CP*TP*CP*TP*C)-3')
L: DNA (5'-D(*GP*AP*GP*AP*GP*AP*AP*GP*CP*GP*CP*CP*AP*AP*AP*CP*AP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)333,45220
Polymers332,19410
Non-polymers1,25810
Water1086
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, homology
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area31120 Å2
ΔGint-262 kcal/mol
Surface area89500 Å2

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Components

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Protein , 2 types, 6 molecules DEABFC

#1: Protein
Integrase /


Mass: 33943.539 Da / Num. of mol.: 4 / Mutation: A219E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Simian T-lymphotropic virus 1 / Gene: pol / Production host: Escherichia coli (E. coli) / Strain (production host): DE3 pLacI (Rosetta-2) / References: UniProt: Q4QY51
#2: Protein PC4 and SFRS1-interacting protein,Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform


Mass: 80394.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: fusion construct containing human LEDGF (residues 1-324 thus without the IBD domain) (gene PSIP1; O75475)) and human B56gammma (residues 11-380) regulatory subunit of PP2A (gene PPP2R5C) (no ...Details: fusion construct containing human LEDGF (residues 1-324 thus without the IBD domain) (gene PSIP1; O75475)) and human B56gammma (residues 11-380) regulatory subunit of PP2A (gene PPP2R5C) (no space for these details below),fusion construct containing human LEDGF (residues 1-324 thus without the IBD domain) (gene PSIP1; O75475)) and human B56gammma (residues 11-380) regulatory subunit of PP2A (gene PPP2R5C) (no space for these details below)
Source: (gene. exp.) Homo sapiens (human) / Gene: PSIP1, DFS70, LEDGF, PSIP2, PPP2R5C, KIAA0044 / Production host: Escherichia coli (E. coli) / Variant (production host): LOBSTR(RIL)

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DNA chain , 2 types, 4 molecules IKJL

#3: DNA chain DNA (5'-D(*AP*CP*TP*GP*TP*GP*TP*TP*TP*GP*GP*CP*GP*CP*TP*TP*CP*TP*CP*TP*C)-3')


Mass: 9221.909 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Simian T-lymphotropic virus 1
#4: DNA chain DNA (5'-D(*GP*AP*GP*AP*GP*AP*AP*GP*CP*GP*CP*CP*AP*AP*AP*CP*AP*CP*A)-3')


Mass: 8593.560 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Simian T-lymphotropic virus 1

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Non-polymers , 4 types, 16 molecules

#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-KLQ / Bictegravir / Bictegravir


Mass: 449.380 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H18F3N3O5 / Feature type: SUBJECT OF INVESTIGATION / Comment: inhibitor*YM
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 6 / 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: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complex of STLV-1 MarB43 integrase with nascent viral DNA, the human PP2A B56 subunit and the drug inhibitor bictegravirCOMPLEXSample composition and source have been described in "macromolecules"#1-#40MULTIPLE SOURCES
2IntegraseCOMPLEX#11RECOMBINANT
3PC4 and SFRS1-interacting protein,Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoformCOMPLEX#21RECOMBINANT
4DNACOMPLEX#3-#41RECOMBINANT
Molecular weightValue: 0.3311 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Simian T-lymphotropic virus 133747
23Homo sapiens (human)9606
34Simian T-lymphotropic virus 133747
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
34Synthetic construct (others)32630
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMBis tris propane1
20.3 MSodium chlorideNaClSodium chloride1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Complex was isolated by size exclusion chromatography
Specimen supportDetails: Glow-discharged for 4 min at 45 mA on an Emitech K100X instrument (Electron Microscopy Sciences) and covered with a layer of graphene oxide (Sigma-Aldrich, catalogue #763705) immediately before being used.
Grid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 295.15 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: dev_4155: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchv0.53particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.12model fitting
9Coot0.8.9.2model refinement
10PHENIXdev-4142model refinement
11cryoSPARCinitial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1714199
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39731 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6Z2Y

6z2y
PDB Unreleased entry


Details: 6Z2Y was fitted into the cryoEM map using Chimera. The model was adjusted to fit the map; metal ions and drug docked into the map manually using Coot. The final model was subjected to Phenix. ...Details: 6Z2Y was fitted into the cryoEM map using Chimera. The model was adjusted to fit the map; metal ions and drug docked into the map manually using Coot. The final model was subjected to Phenix.real_space_refine using C2 NCS and secondary structure and metal ion coordination restraints.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315676
ELECTRON MICROSCOPYf_angle_d0.51621688
ELECTRON MICROSCOPYf_dihedral_angle_d18.3712582
ELECTRON MICROSCOPYf_chiral_restr0.0382412
ELECTRON MICROSCOPYf_plane_restr0.0062464

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