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- PDB-8udk: Human Mitochondrial DNA Polymerase gamma R853A Ternary Complex -

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Basic information

Entry
Database: PDB / ID: 8udk
TitleHuman Mitochondrial DNA Polymerase gamma R853A Ternary Complex
Components
  • (DNA polymerase subunit gamma- ...) x 2
  • DNA (24-MER)
  • DNA (28-MER)
  • DNA (5'-D(P*AP*AP*GP*GP*GP*CP*CP*TP*AP*TP*AP*AP*AP*A)-3')
  • DNA (5'-D(P*TP*TP*TP*TP*AP*TP*AP*GP*GP*CP*CP*CP*TP*T)-3')
KeywordsTRANSFERASE/DNA / Mitochondrial / DNA Polymerase / TRANSFERASE / TRANSFERASE-DNA complex
Function / homology
Function and homology information


gamma DNA polymerase complex / mitochondrial chromosome / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / DNA replication proofreading / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA metabolic process / DNA polymerase processivity factor activity / mitochondrial nucleoid ...gamma DNA polymerase complex / mitochondrial chromosome / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / DNA replication proofreading / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA metabolic process / DNA polymerase processivity factor activity / mitochondrial nucleoid / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / 5'-deoxyribose-5-phosphate lyase activity / DNA polymerase binding / base-excision repair, gap-filling / 3'-5' exonuclease activity / Transcriptional activation of mitochondrial biogenesis / base-excision repair / DNA-templated DNA replication / double-stranded DNA binding / protease binding / in utero embryonic development / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / mitochondrial matrix / intracellular membrane-bounded organelle / chromatin binding / protein-containing complex / mitochondrion / DNA binding / identical protein binding / cytoplasm
Similarity search - Function
DNA-directed DNA-polymerase, family A, mitochondria / DNA mitochondrial polymerase, exonuclease domain / : / DNA mitochondrial polymerase exonuclease domain / POLG2, C-terminal / Glycyl-tRNA synthetase/DNA polymerase subunit gamma-2 / Anticodon-binding / Anticodon binding domain / Anticodon-binding domain superfamily / DNA-directed DNA polymerase, family A, conserved site ...DNA-directed DNA-polymerase, family A, mitochondria / DNA mitochondrial polymerase, exonuclease domain / : / DNA mitochondrial polymerase exonuclease domain / POLG2, C-terminal / Glycyl-tRNA synthetase/DNA polymerase subunit gamma-2 / Anticodon-binding / Anticodon binding domain / Anticodon-binding domain superfamily / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / Class II Aminoacyl-tRNA synthetase/Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL) / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA polymerase subunit gamma-1 / DNA polymerase subunit gamma-2
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.43 Å
AuthorsPark, J. / Herrmann, G.K. / Yin, Y.W.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI134611 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM145925 United States
CitationJournal: Sci Adv / Year: 2024
Title: An interaction network in the polymerase active site is a prerequisite for Watson-Crick base pairing in Pol γ.
Authors: Joon Park / Geoffrey K Herrmann / Arkanil Roy / Christie K Shumate / G Andrés Cisneros / Y Whitney Yin /
Abstract: The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to neurological diseases. Although not ...The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to neurological diseases. Although not a catalytic residue, Pol γ arginine-853 mutants are void of polymerase activity. To identify the structural basis for the disease, we determined a crystal structure of the Pol γ mutant ternary complex with correct incoming nucleotide 2'-deoxycytidine 5'-triphosphate (dCTP). Opposite to the wild type that undergoes open-to-closed conformational changes when bound to a correct nucleotide that is essential for forming a catalytically competent active site, the mutant complex failed to undergo the conformational change, and the dCTP did not base pair with its Watson-Crick complementary templating residue. Our studies revealed that arginine-853 coordinates an interaction network that aligns the 3'-end of primer and dCTP with the catalytic residues. Disruption of the network precludes the formation of Watson-Crick base pairing and closing of the active site, resulting in an inactive polymerase.
History
DepositionSep 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase subunit gamma-1
B: DNA polymerase subunit gamma-2, mitochondrial
C: DNA polymerase subunit gamma-2, mitochondrial
P: DNA (24-MER)
T: DNA (28-MER)
D: DNA (5'-D(P*AP*AP*GP*GP*GP*CP*CP*TP*AP*TP*AP*AP*AP*A)-3')
E: DNA (5'-D(P*TP*TP*TP*TP*AP*TP*AP*GP*GP*CP*CP*CP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)274,5378
Polymers274,0697
Non-polymers4671
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18540 Å2
ΔGint-78 kcal/mol
Surface area84800 Å2
Unit cell
Length a, b, c (Å)215.410, 215.410, 169.966
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Space group name HallP4abw2nw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+1/4
#3: y+1/2,-x+1/2,z+3/4
#4: x+1/2,-y+1/2,-z+3/4
#5: -x+1/2,y+1/2,-z+1/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "B" and (resid 67 through 70 or resid 72...
d_2ens_1(chain "C" and (resid 67 through 70 or resid 72...

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11GLUGLULEULEUBB67 - 7067 - 70
d_12ILEILEVALVALBB72 - 12072 - 120
d_13ARGARGARGARGBB122122
d_14GLNGLNHISHISBB124 - 133124 - 133
d_15LEULEULEULEUBB181 - 190181 - 190
d_16HISHISPHEPHEBB192 - 215192 - 215
d_17PROPROVALVALBB217 - 218217 - 218
d_18LYSLYSLEULEUBB229 - 260229 - 260
d_19TRPTRPASPASPBB262 - 280262 - 280
d_110GLYGLYASPASPBB283 - 308283 - 308
d_111GLUGLUGLYGLYBB310 - 317310 - 317
d_112VALVALGLYGLYBB319 - 324319 - 324
d_113ASPASPASPASPBB326326
d_114ARGARGGLNGLNBB328 - 355328 - 355
d_115LYSLYSLEULEUBB370 - 418370 - 418
d_116THRTHRTHRTHRBB420420
d_117GLNGLNSERSERBB422 - 430422 - 430
d_118TYRTYRVALVALBB432 - 485432 - 485
d_21GLUGLULEULEUCC67 - 7067 - 70
d_22ILEILEVALVALCC72 - 12072 - 120
d_23ARGARGARGARGCC122122
d_24GLNGLNHISHISCC124 - 133124 - 133
d_25LEULEULEULEUCC181 - 190181 - 190
d_26HISHISPHEPHECC192 - 215192 - 215
d_27PROPROVALVALCC217 - 218217 - 218
d_28LYSLYSLEULEUCC229 - 260229 - 260
d_29TRPTRPASPASPCC262 - 280262 - 280
d_210GLYGLYASPASPCC283 - 308283 - 308
d_211GLUGLUGLYGLYCC310 - 317310 - 317
d_212VALVALGLYGLYCC319 - 324319 - 324
d_213ASPASPASPASPCC326326
d_214ARGARGGLNGLNCC328 - 355328 - 355
d_215LYSLYSLEULEUCC370 - 418370 - 418
d_216THRTHRTHRTHRCC420420
d_217GLNGLNVALVALCC422 - 485422 - 485

NCS oper: (Code: givenMatrix: (-0.793421714272, 0.483441954073, -0.369818685795), (0.461787180143, 0.0822866707548, -0.883165615313), (-0.396528162401, -0.871500304565, -0.288535328106)Vector: 127. ...NCS oper: (Code: given
Matrix: (-0.793421714272, 0.483441954073, -0.369818685795), (0.461787180143, 0.0822866707548, -0.883165615313), (-0.396528162401, -0.871500304565, -0.288535328106)
Vector: 127.14376023, 26.8217755099, 101.364703704)

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Components

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DNA polymerase subunit gamma- ... , 2 types, 3 molecules ABC

#1: Protein DNA polymerase subunit gamma-1 / 3'-5' exodeoxyribonuclease / 5'-deoxyribose-phosphate lyase / Mitochondrial DNA polymerase ...3'-5' exodeoxyribonuclease / 5'-deoxyribose-phosphate lyase / Mitochondrial DNA polymerase catalytic subunit / PolG-alpha


Mass: 139542.562 Da / Num. of mol.: 1 / Mutation: D198A, E200A, R853A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLG, MDP1, POLG1, POLGA
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P54098, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters, Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases
#2: Protein DNA polymerase subunit gamma-2, mitochondrial / DNA polymerase gamma accessory 55 kDa subunit / p55 / Mitochondrial DNA polymerase accessory ...DNA polymerase gamma accessory 55 kDa subunit / p55 / Mitochondrial DNA polymerase accessory subunit / MtPolB / PolG-beta


Mass: 54991.000 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLG2, MTPOLB
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: Q9UHN1, DNA-directed DNA polymerase

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DNA chain , 4 types, 4 molecules PTDE

#3: DNA chain DNA (24-MER)


Mass: 7341.773 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (28-MER)


Mass: 8644.536 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain DNA (5'-D(P*AP*AP*GP*GP*GP*CP*CP*TP*AP*TP*AP*AP*AP*A)-3')


Mass: 4321.856 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (5'-D(P*TP*TP*TP*TP*AP*TP*AP*GP*GP*CP*CP*CP*TP*T)-3')


Mass: 4236.763 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 1 types, 1 molecules

#7: Chemical ChemComp-DCP / 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE


Mass: 467.157 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O13P3 / Feature type: SUBJECT OF INVESTIGATION

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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: 3.6 Å3/Da / Density % sol: 65.81 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, hanging drop
Details: 100 mM MES (pH 6.0), 150 mM NaCl, 10 mM CaCl2, 50 mM BME, 3% PEG 8000, 1-8% sucrose, 2% Jeffamine, 0.8 M NDSB

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.979 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Mar 16, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 3.43→50 Å / Num. obs: 5106256 / % possible obs: 100 % / Redundancy: 9.5 % / Biso Wilson estimate: 119.55 Å2 / Rpim(I) all: 0.066 / Net I/σ(I): 15.1
Reflection shellResolution: 3.43→3.48 Å / Num. unique obs: 2869 / Rpim(I) all: 0.53 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.21.1_5286+SVNrefinement
HKL-3000data reduction
HKL-3000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.43→48.87 Å / SU ML: 0.389 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 23.3631
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: The authors state that the DCP ligand has poor density, which is likely the reason for RSR-Z outlier.
RfactorNum. reflection% reflection
Rfree0.2341 2000 3.79 %
Rwork0.1976 50824 -
obs0.199 52824 98.23 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 138.58 Å2
Refinement stepCycle: LAST / Resolution: 3.43→48.87 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13833 1540 28 0 15401
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.002615944
X-RAY DIFFRACTIONf_angle_d0.694321958
X-RAY DIFFRACTIONf_chiral_restr0.03992379
X-RAY DIFFRACTIONf_plane_restr0.00422559
X-RAY DIFFRACTIONf_dihedral_angle_d16.47445994
Refine LS restraints NCSType: Torsion NCS / Rms dev position: 1.08104532628 Å
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.43-3.520.35271330.30293383X-RAY DIFFRACTION93.07
3.52-3.620.29941390.24853527X-RAY DIFFRACTION96.68
3.62-3.720.25511380.23513513X-RAY DIFFRACTION96.74
3.72-3.840.23531390.21223545X-RAY DIFFRACTION97.1
3.84-3.980.26751420.19553572X-RAY DIFFRACTION97.84
3.98-4.140.26031400.19063587X-RAY DIFFRACTION98.49
4.14-4.330.24541440.18973637X-RAY DIFFRACTION98.85
4.33-4.550.21581410.16593612X-RAY DIFFRACTION98.87
4.55-4.840.18221440.15473661X-RAY DIFFRACTION99.35
4.84-5.210.22681440.17613668X-RAY DIFFRACTION99.45
5.21-5.740.23881470.19563693X-RAY DIFFRACTION99.46
5.74-6.560.21711460.21253711X-RAY DIFFRACTION99.54
6.57-8.260.25521480.20923775X-RAY DIFFRACTION99.72
8.27-48.870.21471550.1963940X-RAY DIFFRACTION99.81
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
1-0.1457695369110.1392892968960.1373995528791.408867148070.2356101559910.0606007504247-0.000393405018489-0.05692700293880.02539426165-0.259900038663-0.05025272218720.03119511730770.04680022808780.0492072495921-0.05190979034260.617850586153-0.06285584665230.01625731980140.4955220652930.1668174613530.59826656367753.526-21.37619.054
20.4165727629560.203311150898-0.3266911467081.27202804294-0.163074315970.889819882260.1122044590140.07291655717120.0634343776115-0.133474408669-0.07961186507240.169395656907-0.154278813695-0.00941454391630.6752390150280.05161305933870.06764490868370.5869881481280.09153482978160.55403665239961.77822.72532.039
30.738869681987-0.4527246332540.5200091032021.18686876209-0.3784742520860.880788829017-0.0164311191768-0.2413733283940.1675799632360.4123012811520.0057206435167-0.411011180441-0.3400341623280.07037623776610.05835569043450.84938739926-0.0497366607577-0.06475707478260.6480745512960.0698901377710.78433035495177.17528.96347.434
40.2015017599930.0639426775852-0.0900183278090.187082106333-0.2142149751640.221261302926-0.3759855303170.155277870567-0.587545097545-0.204554685585-0.08355586991440.1187733676950.2980111943520.0503649853407-9.9188974735E-51.83711128969-0.2293807346780.06494042328881.224812545970.1767027870021.4236686183538.612-12.93144.475
50.165260794296-0.277265575321-0.1694591726250.3131816574280.3302884242620.468032063283-0.138634697777-0.39803262976-0.0822992647452-0.3545486437960.8135634702750.287762159891-0.0429818997852-0.01017544831360.03998994321871.544521080730.04704556771890.08349801432041.614665032720.2062867279961.6169980025938.083-15.3440.349
60.147511986657-0.07269999460540.05540381234380.0251742215461-0.02370191777460.0178194675096-0.6737691297180.301871553245-0.837363435242-0.0619050688549-0.1017538534040.0896406225817-0.00735700332950.224229494857-0.000148512882482.269180413080.2640932688950.27779572252.225765242810.353291605782.02673920467104.584-1.78919.793
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN A AND RESID 68:1238 )A68 - 1238
2X-RAY DIFFRACTION2( CHAIN B AND RESID 64:485 )B64 - 485
3X-RAY DIFFRACTION3( CHAIN C AND RESID 67:485 )C67 - 485
4X-RAY DIFFRACTION4( CHAIN P AND ( RESID 3:23 OR RESID 24:24 ) )P3 - 23
5X-RAY DIFFRACTION4( CHAIN P AND ( RESID 3:23 OR RESID 24:24 ) )P24
6X-RAY DIFFRACTION5( CHAIN T AND RESID 2:26 )T2 - 26
7X-RAY DIFFRACTION6( CHAIN D AND RESID 20:33 ) OR ( CHAIN E AND RESID 95:108 )D20 - 33
8X-RAY DIFFRACTION6( CHAIN D AND RESID 20:33 ) OR ( CHAIN E AND RESID 95:108 )E95 - 108

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