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- PDB-6rwo: SIVrcm intasome (Q148H/G140S) in complex with bictegravir -

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Basic information

Entry
Database: PDB / ID: 6rwo
TitleSIVrcm intasome (Q148H/G140S) in complex with bictegravir
Components
  • DNA (5'-D(*AP*AP*CP*TP*GP*GP*TP*AP*GP*AP*GP*AP*TP*TP*TP*TP*TP*CP*TP*TP*AP*GP*C)-3')
  • DNA (5'-D(P*GP*CP*TP*AP*AP*GP*AP*AP*AP*AP*AP*TP*CP*TP*CP*TP*AP*CP*CP*A)-3')
  • Pol protein
KeywordsRECOMBINATION / retroviral integrase / lentivirus / strand transfer inhibior / protein-DNA complex
Function / homology
Function and homology information


exoribonuclease H / exoribonuclease H activity / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / establishment of integrated proviral latency / DNA recombination / viral entry into host cell / aspartic-type endopeptidase activity ...exoribonuclease H / exoribonuclease H activity / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / establishment of integrated proviral latency / DNA recombination / viral entry into host cell / aspartic-type endopeptidase activity / DNA binding / zinc ion binding
Integrase, N-terminal zinc-binding domain / Retropepsins / Reverse transcriptase connection / Integrase-like, N-terminal / Ribonuclease H domain / Peptidase A2A, retrovirus, catalytic / Aspartic peptidase, active site / Integrase, catalytic core / Integrase, C-terminal, retroviral / Reverse transcriptase domain ...Integrase, N-terminal zinc-binding domain / Retropepsins / Reverse transcriptase connection / Integrase-like, N-terminal / Ribonuclease H domain / Peptidase A2A, retrovirus, catalytic / Aspartic peptidase, active site / Integrase, catalytic core / Integrase, C-terminal, retroviral / Reverse transcriptase domain / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Ribonuclease H superfamily / Integrase, C-terminal domain superfamily, retroviral / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily / Integrase DNA binding domain / Integrase core domain / Integrase Zinc binding domain / Reverse transcriptase thumb / Integrase, N-terminal zinc-binding domain / Arc Repressor Mutant, subunit A / Orthogonal Bundle / Mainly Alpha
Exoribonuclease H
Biological speciesSimian immunodeficiency virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsCherepanov, P. / Nans, A. / Cook, N.
Funding support United States, United Kingdom, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM082251 United States
The Francis Crick InstituteFC001061 United Kingdom
CitationJournal: Science / Year: 2020
Title: Structural basis of second-generation HIV integrase inhibitor action and viral resistance.
Authors: Nicola J Cook / Wen Li / Dénes Berta / Magd Badaoui / Allison Ballandras-Colas / Andrea Nans / Abhay Kotecha / Edina Rosta / Alan N Engelman / Peter Cherepanov /
Abstract: Although second-generation HIV integrase strand-transfer inhibitors (INSTIs) are prescribed throughout the world, the mechanistic basis for the superiority of these drugs is poorly understood. We ...Although second-generation HIV integrase strand-transfer inhibitors (INSTIs) are prescribed throughout the world, the mechanistic basis for the superiority of these drugs is poorly understood. We used single-particle cryo-electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near-atomic resolution. Glutamine-148→histidine (Q148H) and glycine-140→serine (G140S) amino acid substitutions in integrase that result in clinical INSTI failure perturb optimal magnesium ion coordination in the enzyme active site. The expanded chemical scaffolds of second-generation compounds mediate interactions with the protein backbone that are critical for antagonizing viruses containing the Q148H and G140S mutations. Our results reveal that binding to magnesium ions underpins a fundamental weakness of the INSTI pharmacophore that is exploited by the virus to engender resistance and provide a structural framework for the development of this class of anti-HIV/AIDS therapeutics.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 5, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2020Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 26, 2020Group: Database references / Structure summary / Category: audit_author / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 18, 2020Group: Database references / Structure summary / Category: pdbx_database_related / struct / Item: _pdbx_database_related.details / _struct.title

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Assembly

Deposited unit
A: Pol protein
Q: DNA (5'-D(*AP*AP*CP*TP*GP*GP*TP*AP*GP*AP*GP*AP*TP*TP*TP*TP*TP*CP*TP*TP*AP*GP*C)-3')
W: DNA (5'-D(P*GP*CP*TP*AP*AP*GP*AP*AP*AP*AP*AP*TP*CP*TP*CP*TP*AP*CP*CP*A)-3')
E: Pol protein
N: Pol protein
L: Pol protein
K: Pol protein
J: Pol protein
I: Pol protein
S: DNA (5'-D(*AP*AP*CP*TP*GP*GP*TP*AP*GP*AP*GP*AP*TP*TP*TP*TP*TP*CP*TP*TP*AP*GP*C)-3')
T: DNA (5'-D(P*GP*CP*TP*AP*AP*GP*AP*AP*AP*AP*AP*TP*CP*TP*CP*TP*AP*CP*CP*A)-3')
M: Pol protein
F: Pol protein
D: Pol protein
C: Pol protein
B: Pol protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)433,57432
Polymers431,98416
Non-polymers1,59016
Water1086
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area49050 Å2
ΔGint-296 kcal/mol
Surface area110940 Å2
MethodPISA

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Components

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Protein , 1 types, 12 molecules AENLKJIMFDCB

#1: Protein
Pol protein


Mass: 32772.223 Da / Num. of mol.: 12 / Mutation: Q148H, G140S, A119D
Source method: isolated from a genetically manipulated source
Details: SIVrcm integrase / Source: (gene. exp.) Simian immunodeficiency virus / Gene: pol / Variant: SIV of red-capped mangabeys / Production host: Escherichia coli K-12 (bacteria) / Variant (production host): PC2 / References: UniProt: E1ANT8

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DNA chain , 2 types, 4 molecules QSWT

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


Mass: 10134.541 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Simian immunodeficiency virus / Gene: U5 LTR end / Variant: SIV of red-capped mangabeys / Production host: synthetic construct (others)
#3: DNA chain DNA (5'-D(P*GP*CP*TP*AP*AP*GP*AP*AP*AP*AP*AP*TP*CP*TP*CP*TP*AP*CP*CP*A)-3')


Mass: 9223.995 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Simian immunodeficiency virus / Gene: U5 LTR end / Variant: SIV of red-capped mangabeys / Production host: synthetic construct (others)

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Non-polymers , 5 types, 22 molecules

#4: Chemical
ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#6: 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: antivirus, inhibitor*YM
#7: Chemical
ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#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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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
IDNameTypeEntity IDParent-IDSource
1SIVrcm intasome in complex with bictegravirCOMPLEX#1-#30MULTIPLE SOURCES
2Pol ProteinCOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Simian immunodeficiency virus11723
23Simian immunodeficiency virus11723
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23synthetic construct (others)32630
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
10.35 Msodium chlorideNaClSodium chloride1
20.025 M4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHepes1
30.005 Mmagnesium sulfateMgSO41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 295 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 / Nominal defocus max: 3500 nm / Nominal defocus min: 1600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6618
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Gautomatch0.56particle selectionparticle picking
2RELION2.1particle selectionparticle picking
3cryoSPARC2particle selection2D classification
4EPUimage acquisition
6Gctf1.06CTF correctionper-particle estimation
9UCSF Chimeramodel fitting
11Cootmodel refinement
12PHENIX1.15.2-3472model refinementphenix.real_space_refine
13cryoSPARC2initial Euler assignment
14cryoSPARC2final Euler assignment
15RELION2.1classification
16cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 217635 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID3D fitting-ID
12B4J1
21EX41
31K6Y1

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