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- PDB-7oz5: Crystal structure of HIV-1 reverse transcriptase with a double st... -

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Entry
Database: PDB / ID: 7oz5
TitleCrystal structure of HIV-1 reverse transcriptase with a double stranded DNA in complex with fragment 166 at the transient P-pocket.
Components
  • DNA (28-MER)
  • DNA (5'-D(*AP*CP*AP*GP*TP*CP*CP*CP*TP*GP*TP*TP*CP*GP*GP*GP*CP*GP*CP*CP*G)-3')
  • Gag-Pol polyprotein
  • Reverse transcriptase/ribonuclease H
KeywordsTRANSFERASE / Reverse Transcriptase / RT-DNA complex / RT sliding / Transferase-DNA complex / P-1 complex / P51 / P66
Function / homology
Function and homology information


HIV-1 retropepsin / 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 ...HIV-1 retropepsin / 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 / host cell / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / symbiont-mediated suppression of host gene expression / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / lipid binding / host cell nucleus / host cell plasma membrane / structural molecule activity / virion membrane / proteolysis / DNA binding / zinc ion binding / membrane
Similarity search - Function
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 ...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
sucrose / Chem-3IR / : / DNA / DNA (> 10) / Gag-Pol polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus type 1 group M subtype B
Human immunodeficiency virus type 1 BH10
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.37 Å
AuthorsMartinez, S.E. / Singh, A.K. / Das, K.
CitationJournal: Nat Commun / Year: 2021
Title: Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - targeting P-pocket by fragment screening.
Authors: Abhimanyu K Singh / Sergio E Martinez / Weijie Gu / Hoai Nguyen / Dominique Schols / Piet Herdewijn / Steven De Jonghe / Kalyan Das /
Abstract: HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the ...HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3'-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized, two of which show noticeable RT inhibition. An engineered RT/DNA aptamer complex could trap the transient P-pocket in solution, and structures of the RT/DNA complex were determined in the presence of an inhibitory fragment. A synthesized analog bound at P-pocket is further analyzed by single-particle cryo-EM. Identification of the P-pocket within HIV RT and the developed structure-based platform provide an opportunity for the design new types of polymerase inhibitors.
History
DepositionJun 25, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 8, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 22, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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.identifier_ORCID
Revision 1.2Jan 31, 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

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Assembly

Deposited unit
A: Reverse transcriptase/ribonuclease H
B: Gag-Pol polyprotein
T: DNA (28-MER)
P: DNA (5'-D(*AP*CP*AP*GP*TP*CP*CP*CP*TP*GP*TP*TP*CP*GP*GP*GP*CP*GP*CP*CP*G)-3')
C: Reverse transcriptase/ribonuclease H
D: Gag-Pol polyprotein
E: DNA (28-MER)
F: DNA (5'-D(*AP*CP*AP*GP*TP*CP*CP*CP*TP*GP*TP*TP*CP*GP*GP*GP*CP*GP*CP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)265,08029
Polymers262,1278
Non-polymers2,95221
Water724
1
A: Reverse transcriptase/ribonuclease H
B: Gag-Pol polyprotein
T: DNA (28-MER)
P: DNA (5'-D(*AP*CP*AP*GP*TP*CP*CP*CP*TP*GP*TP*TP*CP*GP*GP*GP*CP*GP*CP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,41814
Polymers131,0644
Non-polymers1,35410
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14690 Å2
ΔGint-98 kcal/mol
Surface area47960 Å2
MethodPISA
2
C: Reverse transcriptase/ribonuclease H
D: Gag-Pol polyprotein
E: DNA (28-MER)
F: DNA (5'-D(*AP*CP*AP*GP*TP*CP*CP*CP*TP*GP*TP*TP*CP*GP*GP*GP*CP*GP*CP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,66215
Polymers131,0644
Non-polymers1,59811
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13340 Å2
ΔGint-104 kcal/mol
Surface area48690 Å2
MethodPISA
Unit cell
Length a, b, c (Å)310.747, 62.063, 169.292
Angle α, β, γ (deg.)90.000, 104.930, 90.000
Int Tables number5
Space group name H-MC121

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Components

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Protein , 2 types, 4 molecules ACBD

#1: Protein Reverse transcriptase/ribonuclease H / Exoribonuclease H / p66 RT


Mass: 64038.367 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus type 1 group M subtype B (isolate BH10)
Strain: isolate BH10 / Gene: gag-pol / Plasmid: PCDF-2 EK/LIC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): BL21 CodonPLUS RIL
References: UniProt: P03366, RNA-directed DNA polymerase, DNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H
#2: Protein Gag-Pol polyprotein / Pr160Gag-Pol


Mass: 51928.629 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus type 1 BH10
Gene: gag-pol / Plasmid: PCDF-2 EK/LIC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): BL21 codon plus RIL
References: UniProt: P03366, HIV-1 retropepsin, RNA-directed DNA polymerase, DNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H, Transferases; Transferring phosphorus- ...References: UniProt: P03366, HIV-1 retropepsin, RNA-directed DNA polymerase, DNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds

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DNA chain , 2 types, 4 molecules TEPF

#3: DNA chain DNA (28-MER)


Mass: 8680.592 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: DNA TEMPLATE FROM PRIMER BINDING SEQUENCE OF HIV-1 GENOME
Source: (synth.) Human immunodeficiency virus type 1 BH10
#4: DNA chain DNA (5'-D(*AP*CP*AP*GP*TP*CP*CP*CP*TP*GP*TP*TP*CP*GP*GP*GP*CP*GP*CP*CP*G)-3')


Mass: 6416.122 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: DNA SEQUENCE FROM TRNA LYS3 THAT BINDS TO PRIMER BINDING SEQUENCE (PBS) OF HIV-1 GENOME
Source: (synth.) Homo sapiens (human)

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Sugars , 1 types, 2 molecules

#5: Polysaccharide beta-D-fructofuranose-(2-1)-alpha-D-glucopyranose


Type: oligosaccharide, Oligosaccharide / Class: Nutrient / Mass: 342.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: oligosaccharide with reducing-end-to-reducing-end glycosidic bond
References: sucrose
DescriptorTypeProgram
DFrufb2-1DGlcpaGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[ha122h-2b_2-5][a2122h-1a_1-5]/1-2/a2-b1WURCSPDB2Glycan 1.1.0
[][b-D-Fruf]{[(2+1)][a-D-Glcp]{}}LINUCSPDB-CARE

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Non-polymers , 3 types, 23 molecules

#6: Chemical
ChemComp-CD / CADMIUM ION


Mass: 112.411 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Cd
#7: Chemical ChemComp-3IR / (1~{R},2~{R})-2-phenyl-~{N}-(1,3-thiazol-2-yl)cyclopropane-1-carboxamide / ((1R,2R)-2-Phenyl-N-(1,3-thiazol-2-yl) cyclopropanecarboxamide


Mass: 244.312 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C13H12N2OS / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.11 Å3/Da / Density % sol: 60.42 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 11-12% v/v PEG Smear Broad, 10% w/v sucrose, 50 mM PIPES-NaOH pH 6.5, 0.1 M (NH4)2SO4, 5 mM MgCl2, 5 mM CdCl2

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.91589 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Sep 19, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.91589 Å / Relative weight: 1
ReflectionResolution: 3.37→150.13 Å / Num. obs: 44790 / % possible obs: 99.9 % / Redundancy: 5.7 % / Biso Wilson estimate: 98.65 Å2 / CC1/2: 0.987 / Rmerge(I) obs: 0.329 / Rpim(I) all: 0.15 / Rrim(I) all: 0.362 / Net I/σ(I): 3.4
Reflection shellResolution: 3.372→3.43 Å / Redundancy: 5.9 % / Rmerge(I) obs: 2.882 / Num. unique obs: 2220 / CC1/2: 0.338 / Rpim(I) all: 1.296 / Rrim(I) all: 3.168 / % possible all: 100

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Processing

Software
NameVersionClassification
XDSdata reduction
Aimlessdata scaling
PDB_EXTRACT3.27data extraction
PHENIX1.19.1_4122refinement
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6AMO

6amo


Resolution: 3.37→150.13 Å / SU ML: 0.47 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 27.4 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2646 2213 4.99 %
Rwork0.2273 42179 -
obs0.2292 44392 99.03 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 209.95 Å2 / Biso mean: 97.9705 Å2 / Biso min: 30 Å2
Refinement stepCycle: final / Resolution: 3.37→150.13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15830 1782 92 4 17708
Biso mean--102.56 72.36 -
Num. residues----2018
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
3.37-3.450.37211370.3473240691
3.45-3.530.31681230.3166250697
3.53-3.610.34061180.3217265399
3.61-3.710.32281440.30062604100
3.71-3.820.29541260.27522643100
3.82-3.940.32541610.25912616100
3.94-4.090.28891550.2392619100
4.09-4.250.28831530.22382619100
4.25-4.440.25541380.20932688100
4.44-4.680.26581430.20182614100
4.68-4.970.20771470.19832634100
4.97-5.350.25741340.19942680100
5.35-5.890.22761280.20692681100
5.89-6.740.29521290.21952688100
6.74-8.50.20171360.19322716100
8.5-150.130.23781410.2073281299

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