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- PDB-8fnl: Structure of E138K/Q148K HIV-1 intasome with Dolutegravir bound -

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Basic information

Entry
Database: PDB / ID: 8fnl
TitleStructure of E138K/Q148K HIV-1 intasome with Dolutegravir bound
Components
  • DNA (25-MER)
  • DNA (27-MER)
  • Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
KeywordsVIRAL PROTEIN/DNA/INHIBITOR / Integrase / Nucleoprotein complex / Inhibitor / Drug resistance / VIRAL PROTEIN-DNA-INHIBITOR complex
Function / homology
Function and homology information


lamin binding / HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase ...lamin binding / HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / viral penetration into host nucleus / RNA stem-loop binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / nuclear envelope / host cell / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase activity / cadherin binding / symbiont-mediated suppression of host gene expression / viral translational frameshifting / lipid binding / symbiont entry into host cell / chromatin / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / DNA binding / zinc ion binding / nucleus / membrane
Similarity search - Function
Lamina-associated polypeptide 2 alpha, C-terminal / Lamina-associated polypeptide 2 alpha / LEM-like domain / : / Thymopoietin protein / LEM-like domain profile. / Thymopoietin / LEM domain / LEM domain / LEM domain profile. ...Lamina-associated polypeptide 2 alpha, C-terminal / Lamina-associated polypeptide 2 alpha / LEM-like domain / : / Thymopoietin protein / 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. / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / RNase H type-1 domain profile. / Ribonuclease H domain / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Reverse transcriptase domain / Retrovirus capsid, C-terminal / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / 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. / Aspartic peptidase domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Chem-DLU / DNA / DNA (> 10) / Gag-Pol polyprotein / Lamina-associated polypeptide 2, isoform alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Human immunodeficiency virus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsShan, Z.L. / Passos, D.O. / Strutzenberg, T.S. / Li, M. / Lyumkis, D.
Funding support United States, 5items
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 27, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
B: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
C: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
D: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
E: DNA (27-MER)
F: DNA (25-MER)
G: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
H: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
I: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
J: Lamina-associated polypeptide 2, isoform alpha,Integrase chimera
K: DNA (27-MER)
L: DNA (25-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)352,18520
Polymers351,11812
Non-polymers1,0678
Water10,629590
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 8 molecules ABCDGHIJ

#1: Protein
Lamina-associated polypeptide 2, isoform alpha,Integrase chimera / Thymopoietin isoform alpha / TP alpha / Thymopoietin-related peptide isoform alpha / TPRP isoform alpha / IN


Mass: 39899.473 Da / Num. of mol.: 8 / Mutation: E138K,Q148K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Human immunodeficiency virus 1
Gene: TMPO, LAP2, gag-pol / Production host: Escherichia coli (E. coli)
References: UniProt: P42166, UniProt: P12497, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds

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DNA chain , 2 types, 4 molecules EKFL

#2: DNA chain DNA (27-MER)


Mass: 8188.271 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Human immunodeficiency virus 1
#3: DNA chain DNA (25-MER)


Mass: 7773.023 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Human immunodeficiency virus 1

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Non-polymers , 4 types, 598 molecules

#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#6: Chemical 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 / Dolutegravir


Mass: 419.379 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C20H19F2N3O5 / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 590 / 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

ComponentName: E138K/Q148K HIV-1 intasome bound to dolutegravir / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Human immunodeficiency virus 111676
31Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
225 %glycerolC3H8O31
350 mMNDSB-256C12H19NO3S1
45 mMBeta-mercaptoethanolHOCH2CH2SH1
54 micromolarZinc chlorideZnCl21
6100 mMSodium chlorideNaCl1
75 mMCalcium chlorideCaCl21
850 micromolarDolutegravirC20H19F2N3O51
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil
VitrificationCryogen name: ETHANE

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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 FIELD / Calibrated magnification: 57129 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 25 e/Å2 / Detector mode: COUNTING / 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

EM software
IDNameVersionCategory
2Leginonimage acquisition
9PHENIXmodel refinement
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1395715
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 136589 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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