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- PDB-6i4p: Crystal structure of the disease-causing G194C mutant of the huma... -

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Basic information

Entry
Database: PDB / ID: 6i4p
TitleCrystal structure of the disease-causing G194C mutant of the human dihydrolipoamide dehydrogenase
ComponentsDihydrolipoyl dehydrogenase, mitochondrial
KeywordsOXIDOREDUCTASE / Lipoamide dehydrogenase / Pathogenic mutation / E3 deficiency / Alpha-ketoglutarate dehydrogenase complex / 2-oxoglutarate dehydrogenase complex / Pyruvate dehydrogenase complex
Function / homology
Function and homology information


acetyltransferase complex / acrosomal matrix / Glycine degradation / : / dihydrolipoyl dehydrogenase / dihydrolipoyl dehydrogenase activity / acetyl-CoA biosynthetic process from pyruvate / Lysine catabolism / oxoglutarate dehydrogenase complex / : ...acetyltransferase complex / acrosomal matrix / Glycine degradation / : / dihydrolipoyl dehydrogenase / dihydrolipoyl dehydrogenase activity / acetyl-CoA biosynthetic process from pyruvate / Lysine catabolism / oxoglutarate dehydrogenase complex / : / branched-chain amino acid catabolic process / Citric acid cycle (TCA cycle) / pyruvate dehydrogenase complex / Branched-chain amino acid catabolism / Pyruvate metabolism / Glyoxylate metabolism and glycine degradation / Regulation of pyruvate dehydrogenase (PDH) complex / motile cilium / sperm capacitation / Signaling by Retinoic Acid / mitochondrial electron transport, NADH to ubiquinone / gastrulation / Mitochondrial protein degradation / regulation of membrane potential / flavin adenine dinucleotide binding / mitochondrial matrix / mitochondrion / proteolysis / nucleus
Similarity search - Function
Dihydrolipoamide dehydrogenase / Pyridine nucleotide-disulphide oxidoreductase, class I / FAD/NAD-linked reductase, C-terminal dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, class I, active site / Pyridine nucleotide-disulphide oxidoreductases class-I active site. / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / FAD/NAD-linked reductase, dimerisation domain superfamily / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase ...Dihydrolipoamide dehydrogenase / Pyridine nucleotide-disulphide oxidoreductase, class I / FAD/NAD-linked reductase, C-terminal dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, class I, active site / Pyridine nucleotide-disulphide oxidoreductases class-I active site. / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / FAD/NAD-linked reductase, dimerisation domain superfamily / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase / Enolase-like; domain 1 / FAD/NAD(P)-binding domain / FAD/NAD(P)-binding domain / 3-Layer(bba) Sandwich / FAD/NAD(P)-binding domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / Dihydrolipoyl dehydrogenase, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.6 Å
AuthorsSzabo, E. / Wilk, P. / Torocsik, B. / Weiss, M.S. / Adam-Vizi, V. / Ambrus, A.
Funding support Hungary, United States, Germany, 10items
OrganizationGrant numberCountry
Hungarian Academy of Sciences02001 [to A.-V.V.] Hungary
Hungarian Scientific Research Fund, grant#112230 [to A.-V.V.] Hungary
Hungarian Brain Research Pogram, grant#KTIA_13_NAP_III/6 and 2017-1.2.1-NKP-2017-00002 [to A.-V.V.] Hungary
Hungarian Academy of SciencesBolyai Fellowship [to A.A.] Hungary
European Molecular Biology OrganizationShort-term Fellowship [to A.A.] Hungary
Semmelweis University, Young Investigator Research Grant [to A.A.] Hungary
Gedeon Richter PIc., Young Investigator Research Grant [to A.A.] Hungary
Fulbright Commission, Fulbright Fellowship [to A.A.] United States
European Union and Government of Hungary, grant#EFOP-3.6.3-VEKOP-16-2017-00009 [to S.E.] Hungary
European Union CALIPSOplus, grant#16204087-ST [to S.E. and A.A.] Germany
CitationJournal: Hum.Mol.Genet. / Year: 2019
Title: Underlying molecular alterations in human dihydrolipoamide dehydrogenase deficiency revealed by structural analyses of disease-causing enzyme variants.
Authors: Szabo, E. / Wilk, P. / Nagy, B. / Zambo, Z. / Bui, D. / Weichsel, A. / Arjunan, P. / Torocsik, B. / Hubert, A. / Furey, W. / Montfort, W.R. / Jordan, F. / Weiss, M.S. / Adam-Vizi, V. / Ambrus, A.
History
DepositionNov 10, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 20, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dihydrolipoyl dehydrogenase, mitochondrial
B: Dihydrolipoyl dehydrogenase, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,22115
Polymers105,3672
Non-polymers2,85413
Water10,575587
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13150 Å2
ΔGint-162 kcal/mol
Surface area35420 Å2
MethodPISA
Unit cell
Length a, b, c (Å)119.487, 169.423, 61.865
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Components on special symmetry positions
IDModelComponents
11A-857-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 3 through 44 or resid 46...
21(chain B and (resid 3 through 44 or resid 46...

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and (resid 3 through 44 or resid 46...A3 - 44
121(chain A and (resid 3 through 44 or resid 46...A46 - 49
131(chain A and (resid 3 through 44 or resid 46...A51 - 57
141(chain A and (resid 3 through 44 or resid 46...A106 - 139
151(chain A and (resid 3 through 44 or resid 46...A3 - 474
161(chain A and (resid 3 through 44 or resid 46...A141 - 193
171(chain A and (resid 3 through 44 or resid 46...A3 - 474
181(chain A and (resid 3 through 44 or resid 46...A221 - 227
191(chain A and (resid 3 through 44 or resid 46...A3 - 474
1101(chain A and (resid 3 through 44 or resid 46...A278 - 33
1111(chain A and (resid 3 through 44 or resid 46...A353 - 9
1121(chain A and (resid 3 through 44 or resid 46...A353 - 396
1131(chain A and (resid 3 through 44 or resid 46...A398 - 413
1141(chain A and (resid 3 through 44 or resid 46...A446 - 47444
1151(chain A and (resid 3 through 44 or resid 46...A446 - 470
1161(chain A and (resid 3 through 44 or resid 46...A472 - 474
211(chain B and (resid 3 through 44 or resid 46...B3 - 44
221(chain B and (resid 3 through 44 or resid 46...B46 - 49
231(chain B and (resid 3 through 44 or resid 46...B51 - 57
241(chain B and (resid 3 through 44 or resid 46...B59 - 64
251(chain B and (resid 3 through 44 or resid 46...B196 - 219
261(chain B and (resid 3 through 44 or resid 46...B221 - 227
271(chain B and (resid 3 through 44 or resid 46...B229 - 276
281(chain B and (resid 3 through 44 or resid 46...B278 - 331
291(chain B and (resid 3 through 44 or resid 46...B333 - 347
2101(chain B and (resid 3 through 44 or resid 46...B349 - 351
2111(chain B and (resid 3 through 44 or resid 46...B353 - 39436
2121(chain B and (resid 3 through 44 or resid 46...B43813
2131(chain B and (resid 3 through 44 or resid 46...B415 - 436
2141(chain B and (resid 3 through 44 or resid 46...B438 - 444
2151(chain B and (resid 3 through 44 or resid 46...B446 - 470
2161(chain B and (resid 3 through 44 or resid 46...B472 - 474

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Components

#1: Protein Dihydrolipoyl dehydrogenase, mitochondrial / Dihydrolipoamide dehydrogenase / Glycine cleavage system L protein


Mass: 52683.262 Da / Num. of mol.: 2 / Mutation: G194C
Source method: isolated from a genetically manipulated source
Details: Sequence of the Strep-tag with the linker amino acids: MASWSHPQFEKGALEVLFQGPG
Source: (gene. exp.) Homo sapiens (human) / Gene: DLD, GCSL, LAD, PHE3 / Plasmid: pET52b+ / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P09622, dihydrolipoyl dehydrogenase
#2: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#3: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-BTB / 2-[BIS-(2-HYDROXY-ETHYL)-AMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / BIS-TRIS BUFFER


Mass: 209.240 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H19NO5 / Comment: pH buffer*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 587 / Source method: isolated from a natural source / Formula: H2O

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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: 3.07 Å3/Da / Density % sol: 59.94 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.9
Details: 2 M ammonium sulfate, 2(v/v)% PEG 400, 0.1 M Bis-Tris (pH 6.9)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.9184 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Aug 25, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 1.6→46.093 Å / Num. obs: 162966 / % possible obs: 97.4 % / Redundancy: 6.452 % / Biso Wilson estimate: 26.65 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.082 / Rrim(I) all: 0.089 / Χ2: 1.355 / Net I/σ(I): 11.26
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
1.6-1.695.9391.9620.57238410.6692.15189.1
1.69-1.816.8270.941.31251880.9051.018100
1.81-1.956.5050.5942.31230200.9570.64698
1.95-2.146.8830.2565.18217070.9910.277100
2.14-2.396.3950.1649.25189770.9940.17996.7
2.39-2.766.7720.09916.03173740.9970.10799.9
2.76-3.386.2860.0627.09147170.9980.06599.3
3.38-4.775.8330.04140.29115190.9990.04599.2
4.77-46.0936.0480.03246.166230.9990.03598.9

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Phasing

PhasingMethod: molecular replacement
Phasing MRR rigid body: 0.64
Highest resolutionLowest resolution
Rotation48.82 Å2 Å

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Processing

Software
NameVersionClassification
MxCuBEdata collection
XDSdata reduction
XSCALEdata scaling
MOLREPphasing
PHENIXrefinement
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1ZMD

1zmd


Resolution: 1.6→46.093 Å / SU ML: 0.25 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 31.05
Details: TLS groups, NCS torsion-angle restraints, automatic occupancy refinement and real-space refinement were involved, hydrogen atoms were added to the model during refinement
RfactorNum. reflection% reflection
Rfree0.2115 2086 1.29 %
Rwork0.1873 --
obs0.1876 161663 96.64 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 221.66 Å2 / Biso mean: 47.0065 Å2 / Biso min: 20.33 Å2
Refinement stepCycle: final / Resolution: 1.6→46.093 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7081 0 279 587 7947
Biso mean--47.14 43.15 -
Num. residues----953
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4024X-RAY DIFFRACTION6.43TORSIONAL
12B4024X-RAY DIFFRACTION6.43TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 15

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.5956-1.63270.51151040.49457967807173
1.6327-1.67350.46011330.4323102441037794
1.6735-1.71880.34971420.3599108461098899
1.7188-1.76930.31341410.299108051094699
1.7693-1.82650.32111410.2716108091095099
1.8265-1.89170.2711410.2342107631090499
1.8917-1.96750.23741370.2923104151055295
1.9675-2.0570.25461430.2045109201106399
2.057-2.16550.24031420.18751090811050100
2.1655-2.30110.26151370.226104101054795
2.3011-2.47880.20751420.1672109301107299
2.4788-2.72820.21921440.16921099711141100
2.7282-3.12290.21661440.1766110091115399
3.1229-3.93420.17441450.16061112411269100
3.9342-46.11260.15551500.1447114301158099
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)
10.93440.38520.29472.2185-0.05690.51340.0174-0.05390.21050.14640.02630.3397-0.0231-0.0921-0.02550.27810.02140.10860.23010.00550.324631.047210.611620.5549
21.1838-0.9030.48483.2573-0.65520.81660.07160.20040.2546-0.82350.05740.33610.146-0.077-0.11480.5158-0.0375-0.04970.29270.04590.300428.57242.5678-2.4627
32.61430.28211.22422.0468-0.05751.3810.0230.16110.1266-0.33040.0783-0.0225-0.00030.0234-0.03050.3553-0.01150.09820.22540.06340.305840.551317.12776.071
40.92230.65790.39912.23840.08180.4721-0.01590.0927-0.1282-0.25720.0928-0.4140.00710.0431-0.07180.2855-0.00490.10790.2195-0.01980.308646.4675-8.915512.1527
51.4552-0.7646-0.29032.4660.16510.65720.0440.1624-0.186-0.32270.060.16840.1107-0.0296-0.08890.349-0.0372-0.02520.2463-0.02080.341634.2223-37.358312.0963
61.3033-0.3028-0.08362.6753-0.07420.09590.09730.1185-0.1529-0.29490.04580.64850.0574-0.0578-0.08670.3235-0.0181-0.09220.31160.07260.448220.1727-23.551612.5956
71.10850.3265-0.18262.82520.44342.03040.04880.2134-0.378-0.29740.0864-0.18530.31030.1123-0.12730.47680.0195-0.04450.2858-0.07440.504140.7002-52.014212.2461
81.64050.06660.29033.6459-0.66771.73920.0497-0.2202-0.14180.36950.08330.58990.054-0.0747-0.12410.3389-0.05320.06950.25640.07140.507717.3636-37.527529.7487
91.20740.9832-0.09831.2915-0.4021.23940.1909-0.2412-0.08920.56090.00350.4581-0.0372-0.1106-0.1640.4748-0.03510.17530.27930.05670.435821.7627-28.001937.7412
102.5330.2683-0.29351.6918-0.18651.44260.0929-0.3632-0.35270.32180.04460.4953-0.0332-0.0820.01190.4353-0.05150.08710.2910.09610.448124.1612-40.30435.1515
112.1763-0.8506-0.82452.64190.55531.83370.0013-0.1663-0.10190.28910.1998-0.59460.19770.2555-0.17020.30290.0197-0.09840.2655-0.0540.425847.4852-39.755924.03
121.44230.1082-0.28533.9495-0.2240.85550.0957-0.1849-0.00480.5462-0.0432-0.2782-0.03090.0165-0.04940.3562-0.02110.00860.2132-0.00680.247642.0419-11.413730.7792
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 3 through 187 )A3 - 187
2X-RAY DIFFRACTION2chain 'A' and (resid 188 through 260 )A188 - 260
3X-RAY DIFFRACTION3chain 'A' and (resid 261 through 307 )A261 - 307
4X-RAY DIFFRACTION4chain 'A' and (resid 308 through 474 )A308 - 474
5X-RAY DIFFRACTION5chain 'B' and (resid -6 through 67 )B-6 - 67
6X-RAY DIFFRACTION6chain 'B' and (resid 68 through 110 )B68 - 110
7X-RAY DIFFRACTION7chain 'B' and (resid 111 through 152 )B111 - 152
8X-RAY DIFFRACTION8chain 'B' and (resid 153 through 187 )B153 - 187
9X-RAY DIFFRACTION9chain 'B' and (resid 188 through 260 )B188 - 260
10X-RAY DIFFRACTION10chain 'B' and (resid 261 through 288 )B261 - 288
11X-RAY DIFFRACTION11chain 'B' and (resid 289 through 358 )B289 - 358
12X-RAY DIFFRACTION12chain 'B' and (resid 359 through 474 )B359 - 474

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