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- EMDB-29321: Structure of E138K/G140S/Q148H HIV-1 intasome with Dolutegravir bound -

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Basic information

Entry
Database: EMDB / ID: EMD-29321
TitleStructure of E138K/G140S/Q148H HIV-1 intasome with Dolutegravir bound
Map dataDensity modified and resampled map of E138K/G140S/Q148H HIV-1 intasome with drug Dolutegravir
Sample
  • Complex: E138K/G140S/Q148H HIV-1 intasome bound to dolutegravir
    • Protein or peptide: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
    • DNA: DNA (27-MER)
    • DNA: DNA (25-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide
  • Ligand: water
KeywordsIntegrase / Nucleoprotein complex / Inhibitor / Drug resistance / VIRAL PROTEIN-DNA-INHIBITOR complex
Function / homology
Function and homology information


lamin binding / : / : / HIV-1 retropepsin / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / RNA-directed DNA polymerase ...lamin binding / : / : / HIV-1 retropepsin / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / viral penetration into host nucleus / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / nuclear envelope / symbiont-mediated suppression of host gene expression / viral nucleocapsid / DNA recombination / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / cadherin binding / lipid binding / host cell nucleus / chromatin / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / DNA binding / RNA binding / zinc ion binding / membrane / nucleus
Similarity search - Function
LEM-like domain / Lamina-associated polypeptide 2 alpha, C-terminal / Thymopoietin protein / Lamina-associated polypeptide 2 alpha / LEM-like domain profile. / Thymopoietin / LEM domain / LEM domain / LEM domain profile. / in nuclear membrane-associated proteins ...LEM-like domain / Lamina-associated polypeptide 2 alpha, C-terminal / Thymopoietin protein / Lamina-associated polypeptide 2 alpha / LEM-like domain profile. / Thymopoietin / LEM domain / LEM domain / LEM domain profile. / in nuclear membrane-associated proteins / LEM/LEM-like domain superfamily / Reverse transcriptase connection / Reverse transcriptase connection domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain / Integrase, C-terminal, retroviral / Integrase DNA binding domain profile. / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Ribonuclease H domain / RNase H type-1 domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Retrovirus capsid, C-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Gag-Pol polyprotein / Lamina-associated polypeptide 2, isoform alpha
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsShan ZL / Passos DO / Strutzenberg TS / Li M / Lyumkis D
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U01 AI136680 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI146017 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U54 AI170855 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM132090 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM148049 United States
CitationJournal: Sci Adv / Year: 2023
Title: Mechanisms of HIV-1 integrase resistance to dolutegravir and potent inhibition of drug-resistant variants.
Authors: Min Li / Dario Oliveira Passos / Zelin Shan / Steven J Smith / Qinfang Sun / Avik Biswas / Indrani Choudhuri / Timothy S Strutzenberg / Allan Haldane / Nanjie Deng / Zhaoyang Li / Xue Zhi ...Authors: Min Li / Dario Oliveira Passos / Zelin Shan / Steven J Smith / Qinfang Sun / Avik Biswas / Indrani Choudhuri / Timothy S Strutzenberg / Allan Haldane / Nanjie Deng / Zhaoyang Li / Xue Zhi Zhao / Lorenzo Briganti / Mamuka Kvaratskhelia / Terrence R Burke / Ronald M Levy / Stephen H Hughes / Robert Craigie / Dmitry Lyumkis /
Abstract: HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), ...HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.
History
DepositionDec 28, 2022-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29321.png

Downloads & links

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Map

FileDownload / File: emd_29321.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity modified and resampled map of E138K/G140S/Q148H HIV-1 intasome with drug Dolutegravir
Voxel sizeX=Y=Z: 0.38 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-5.045301 - 9.863977999999999
Average (Standard dev.)0.0012824219 (±0.33317348)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 121.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29321_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map of E138K/G140S/Q148H HIV-1 intasome with drug...

Fileemd_29321_additional_1.map
AnnotationUnsharpened map of E138K/G140S/Q148H HIV-1 intasome with drug Dolutegravir
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map1 of E138K/G140S/Q148H HIV-1 intasome with drug Dolutegravir

Fileemd_29321_half_map_1.map
AnnotationHalf map1 of E138K/G140S/Q148H HIV-1 intasome with drug Dolutegravir
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map2 of E138K/G140S/Q148H HIV-1 intasome with drug Dolutegravir

Fileemd_29321_half_map_2.map
AnnotationHalf map2 of E138K/G140S/Q148H HIV-1 intasome with drug Dolutegravir
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E138K/G140S/Q148H HIV-1 intasome bound to dolutegravir

EntireName: E138K/G140S/Q148H HIV-1 intasome bound to dolutegravir
Components
  • Complex: E138K/G140S/Q148H HIV-1 intasome bound to dolutegravir
    • Protein or peptide: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
    • DNA: DNA (27-MER)
    • DNA: DNA (25-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide
  • Ligand: water

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Supramolecule #1: E138K/G140S/Q148H HIV-1 intasome bound to dolutegravir

SupramoleculeName: E138K/G140S/Q148H HIV-1 intasome bound to dolutegravir
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera

MacromoleculeName: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 39.938465 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSHMPKRGRP AATEVKIPKP RGRPPLPAGT NSKGPPDFSS DEEREPTPVL GSGAAAAGQS RAAVGRKATK KTDGGGFLDG IDKAQEEHE KYHSNWRAMA SDFNLPPVVA KEIVASCDKC QLKGEAMHGQ VDCSPGIWQL DCTHLEGKVI LVAVHVASGY I EAEVIPAE ...String:
GSHMPKRGRP AATEVKIPKP RGRPPLPAGT NSKGPPDFSS DEEREPTPVL GSGAAAAGQS RAAVGRKATK KTDGGGFLDG IDKAQEEHE KYHSNWRAMA SDFNLPPVVA KEIVASCDKC QLKGEAMHGQ VDCSPGIWQL DCTHLEGKVI LVAVHVASGY I EAEVIPAE TGQETAYFLL KLAGRWPVKT VHTDNGSNFT STTVKAACWW AGIKQKFSIP YNPQSHGVIE SMNKELKKII GQ VRDQAEH LKTAVQMAVF IHNFKRKGGI GGYSAGERIV DIIATDIQTK ELQKQITKIQ NFRVYYRDSR DPVWKGPAKL LWK GEGAVV IQDNSDIKVV PRRKAKIIRD YGKQMAGDDC VASRQDED

UniProtKB: Lamina-associated polypeptide 2, isoform alpha, Gag-Pol polyprotein

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Macromolecule #2: DNA (27-MER)

MacromoleculeName: DNA (27-MER) / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 8.188271 KDa
SequenceString:
(DA)(DC)(DT)(DG)(DC)(DT)(DA)(DG)(DA)(DG) (DA)(DT)(DT)(DT)(DT)(DC)(DC)(DC)(DG)(DC) (DC)(DC)(DA)(DC)(DG)(DC)(DT)

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Macromolecule #3: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 7.773023 KDa
SequenceString:
(DA)(DG)(DC)(DG)(DT)(DG)(DG)(DG)(DC)(DG) (DG)(DG)(DA)(DA)(DA)(DA)(DT)(DC)(DT)(DC) (DT)(DA)(DG)(DC)(DA)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4...

MacromoleculeName: (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide
type: ligand / ID: 6 / Number of copies: 2 / Formula: DLU
Molecular weightTheoretical: 419.379 Da
Chemical component information

ChemComp-DLU:
(4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide / medication, antiretroviral*YM / Dolutegravir

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 822 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
25.0 %C3H8O3glycerol
50.0 mMC12H19NO3SNDSB-256
5.0 mMHOCH2CH2SHBeta-mercaptoethanol2-Mercaptoethanol
4.0 micromolarZnCl2Zinc chloride
100.0 mMNaClSodium chlorideSodium chloride
5.0 mMCaCl2Calcium chloride
50.0 micromolarC20H19F2N3O5Dolutegravir
GridModel: Quantifoil / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 60240 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1146962
Startup modelType of model: OTHER / Details: Published structure EMD-21151
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 440567

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8fnp:
Structure of E138K/G140S/Q148H HIV-1 intasome with Dolutegravir bound

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