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Basic information

Entry
Database: PDB / ID: 1wci
TitleReactivity modulation of human branched-chain alpha-ketoacid dehydrogenase by an internal molecular switch
Components(2-OXOISOVALERATE DEHYDROGENASE ...) x 2
KeywordsOXIDOREDUCTASE / KETOACID DEHYDROGENASE / BRANCHED-CHAIN / MULTI ENZYME COMPLEX / ACYLATION / OXIDATIVE DECARBOXYLATION / MAPLE SYRUP URINE DISEASE / THIAMINE DIPHOSPHATE / PHOSPHORYLATION / CONFORMATIONAL SWITCH / REACTIVITY / FLAVOPROTEIN / MAPLE SYRU URINE DISEASE / MITOCHONDRION / THIAMINE
Function / homology
Function and homology information


3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring) / 3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring) activity / branched-chain alpha-ketoacid dehydrogenase complex / BCKDH synthesizes BCAA-CoA from KIC, KMVA, KIV / oxoglutarate dehydrogenase complex / branched-chain amino acid catabolic process / Branched-chain amino acid catabolism / carboxy-lyase activity / response to glucocorticoid / response to cAMP ...3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring) / 3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring) activity / branched-chain alpha-ketoacid dehydrogenase complex / BCKDH synthesizes BCAA-CoA from KIC, KMVA, KIV / oxoglutarate dehydrogenase complex / branched-chain amino acid catabolic process / Branched-chain amino acid catabolism / carboxy-lyase activity / response to glucocorticoid / response to cAMP / response to nutrient / lipid metabolic process / mitochondrial matrix / protein-containing complex binding / nucleolus / mitochondrion / nucleoplasm / metal ion binding
Similarity search - Function
: / Transketolase, C-terminal domain / Dehydrogenase, E1 component / Dehydrogenase E1 component / Transketolase, C-terminal domain / Rossmann fold - #920 / Transketolase-like, pyrimidine-binding domain / Transketolase, pyrimidine binding domain / Transketolase, pyrimidine binding domain / Transketolase C-terminal/Pyruvate-ferredoxin oxidoreductase domain II ...: / Transketolase, C-terminal domain / Dehydrogenase, E1 component / Dehydrogenase E1 component / Transketolase, C-terminal domain / Rossmann fold - #920 / Transketolase-like, pyrimidine-binding domain / Transketolase, pyrimidine binding domain / Transketolase, pyrimidine binding domain / Transketolase C-terminal/Pyruvate-ferredoxin oxidoreductase domain II / Thiamin diphosphate (ThDP)-binding fold, Pyr/PP domains / Thiamin diphosphate-binding fold / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
: / : / C2-1-HYDROXY-3-METHYL-BUTYL-THIAMIN / 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial / 2-oxoisovalerate dehydrogenase subunit beta, mitochondrial
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.84 Å
AuthorsMachius, M. / Wynn, R.M. / Chuang, J.L. / Tomchick, D.R. / Brautigam, C.A. / Chuang, D.T.
Citation
Journal: Structure / Year: 2006
Title: A Versatile Conformational Switch Regulates Reactivity in Human Branched-Chain Alpha-Ketoacid Dehydrogenase.
Authors: Machius, M. / Wynn, R.M. / Chuang, J.L. / Li, J. / Kluger, R. / Yu, D. / Tomchick, D.R. / Brautigam, C.A. / Chuang, D.T.
#1: Journal: J.Biol.Chem. / Year: 2004
Title: Crosstalk between Cofactor Binding and the Phosphorylation Loop Conformation in the Bckd Machine
Authors: Li, J. / Wynn, R.M. / Machius, M. / Chuang, J.L. / Karthikeyan, S. / Tomchick, D.R. / Chuang, D.T.
#2: Journal: J.Biol.Chem. / Year: 2003
Title: Roles of His291-Alpha and His146-Beta in the Reductive Acylation Reaction Catalyzed by Human Branched-Chain Alpha-Ketoacid Dehydrogenase: Refined Phosphorylation Loop Structure in the Active Site
Authors: Wynn, R. / Machius, M. / Chuang, J. / Li, J. / Tomchick, D. / Chuang, D.
#3: Journal: J.Biol.Chem. / Year: 2001
Title: Roles of Active Site and Novel K+ Ion-Binding Site Residues in Human Mitochondrial Branched-Chain Alpha-Ketoacid Decarboxylase/Dehydrogenase
Authors: Wynn, R.M. / Ho, R. / Chuang, J.L. / Chuang, D.T.
History
DepositionNov 16, 2004Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 16, 2006Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3May 15, 2019Group: Data collection / Experimental preparation / Other
Category: exptl_crystal_grow / pdbx_database_proc / pdbx_database_status
Item: _exptl_crystal_grow.temp / _pdbx_database_status.recvd_author_approval
Revision 1.4Sep 25, 2019Group: Data collection / Experimental preparation / Other / Category: exptl_crystal_grow / pdbx_database_status
Item: _exptl_crystal_grow.method / _pdbx_database_status.status_code_sf
Revision 1.5Dec 13, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 2-OXOISOVALERATE DEHYDROGENASE ALPHA SUBUNIT
B: 2-OXOISOVALERATE DEHYDROGENASE BETA SUBUNIT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)84,37310
Polymers83,4732
Non-polymers9008
Water10,899605
1
A: 2-OXOISOVALERATE DEHYDROGENASE ALPHA SUBUNIT
B: 2-OXOISOVALERATE DEHYDROGENASE BETA SUBUNIT
hetero molecules

A: 2-OXOISOVALERATE DEHYDROGENASE ALPHA SUBUNIT
B: 2-OXOISOVALERATE DEHYDROGENASE BETA SUBUNIT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)168,74620
Polymers166,9474
Non-polymers1,79916
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation5_555x-y,-y,-z+2/31
MethodPQS
Unit cell
Length a, b, c (Å)145.044, 145.044, 69.510
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number152
Space group name H-MP3121
Components on special symmetry positions
IDModelComponents
11B-2100-

HOH

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Components

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2-OXOISOVALERATE DEHYDROGENASE ... , 2 types, 2 molecules AB

#1: Protein 2-OXOISOVALERATE DEHYDROGENASE ALPHA SUBUNIT / MITOCHONDRIAL PRECURSOR / BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE E1 COMPONENT ALPHA CHAIN / ...MITOCHONDRIAL PRECURSOR / BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE E1 COMPONENT ALPHA CHAIN / BCKDH E1-ALPHA / BCKDE1A


Mass: 45571.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human)
Description: BL-21 CELLS WITH OVEREXPRESSING GROEL AND GROES
Plasmid: PTRC-ALPHA-BETAHIS / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: P12694, 3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring)
#2: Protein 2-OXOISOVALERATE DEHYDROGENASE BETA SUBUNIT / MITOCHONDRIAL PRECURSOR / BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE E1 COMPONENT BETA CHAIN / BCKDH E1-BETA


Mass: 37902.270 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human)
Description: BL-21 CELLS WITH OVEREXPRESSING GROEL AND GROES
Plasmid: PTRC-ALPHA-BETAHIS / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: P21953, 3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring)

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Non-polymers , 6 types, 613 molecules

#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-WWF / C2-1-HYDROXY-3-METHYL-BUTYL-THIAMIN


Mass: 511.447 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H29N4O8P2S
#5: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
#6: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn
#7: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 605 / Source method: isolated from a natural source / Formula: H2O

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Details

Compound detailsCATALYTIC ACTIVITY: 3-METHYL-2-OXOBUTANOATE + [DIHYDROLIPOYLLYSINE-RESIDUE (2-METHYLPROPANOYL) ...CATALYTIC ACTIVITY: 3-METHYL-2-OXOBUTANOATE + [DIHYDROLIPOYLLYSINE-RESIDUE (2-METHYLPROPANOYL)TRANSFERASE] LIPOYLLYSINE = [DIHYDROLIPOYLLYSINE-RESIDUE (2- METHYLPROPANOYL)TRANSFERASE] S-(2- METHYLPROPANOYL)DIHYDROLIPOYLLYSINE + CO(2).

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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: 2.5 Å3/Da / Density % sol: 53.4 %
Crystal growTemperature: 293 K / Method: vapor diffusion / pH: 5.5
Details: CRYSTALS WERE GROWN AT 20C VIA THE VAPOR DIFFUSION METHOD BY MIXING EQUAL AMOUNTS OF PROTEIN (20-25 MG/ML IN 50 MM HEPES/NAOH, PH 7.5, 250 MM KCL, 0.5 MM PMSF, 1 MM BENZAMIDINE AND 5% (V/V) ...Details: CRYSTALS WERE GROWN AT 20C VIA THE VAPOR DIFFUSION METHOD BY MIXING EQUAL AMOUNTS OF PROTEIN (20-25 MG/ML IN 50 MM HEPES/NAOH, PH 7.5, 250 MM KCL, 0.5 MM PMSF, 1 MM BENZAMIDINE AND 5% (V/V) GLYCEROL) WITH WELL SOLUTION (1.4-1.6 M AMMONIUM SULFATE, 0.1 M NA-CITRATE PH 5.8, 20 MM B-MERCAPTOETHANOL). SERIALLY DILUTED CRUSHED CRYSTALS WERE USED FOR MICRO-SEEDING ONE DAY AFTER THE DROPS WERE SET UP. CRYSTALS APPEARED ONE DAY AFTER SEEDING AND GREW TO A MAXIMUM SIZE OF 120 X 800 UM WITHIN 10 DAYS. CRYSTALS WERE STABILIZED FOR 12 HOURS BY TRANSFER TO FRESH WELL SOLUTION. THEY WERE THEN CRYO-PROTECTED BY STEP-WISE TRANSFER INTO CRYO-BUFFER CONTAINING 1.6 M AMMONIUM SULFATE, 50 MM HEPES, PH 7.5, 100 MM NA-CITRATE, PH 5.8, 100 MM KCL, 50 MM DTT AND UP TO 20% (V/V) GLYCEROL. IT WAS FOUND THAT MN2 IONS COULD REPLACE THE MG2 REQUIRED FOR THE BINDING OF THDP TO THE ENZYME.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-BM / Wavelength: 1.00691
DetectorType: CUSTOM / Detector: CCD / Date: Jun 21, 2003
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.00691 Å / Relative weight: 1
ReflectionResolution: 1.84→22.83 Å / Num. obs: 71058 / % possible obs: 97.3 % / Observed criterion σ(I): -3 / Redundancy: 6 % / Biso Wilson estimate: 18.23 Å2 / Rmerge(I) obs: 0.08 / Net I/σ(I): 22.5
Reflection shellResolution: 1.84→1.87 Å / Redundancy: 5.2 % / Rmerge(I) obs: 0.8 / Mean I/σ(I) obs: 1.9 / % possible all: 80.8

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Processing

Software
NameVersionClassification
REFMAC5.2.0003refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1OLS

1ols


Resolution: 1.84→30 Å / Cor.coef. Fo:Fc: 0.967 / Cor.coef. Fo:Fc free: 0.948 / SU B: 4.229 / SU ML: 0.067 / Cross valid method: THROUGHOUT / ESU R: 0.107 / ESU R Free: 0.107 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.188 1477 2.1 %RANDOM
Rwork0.148 ---
obs0.149 69564 97.9 %-
Displacement parametersBiso mean: 18.29 Å2
Baniso -1Baniso -2Baniso -3
1-0.79 Å20.39 Å20 Å2
2--0.79 Å20 Å2
3----1.18 Å2
Refinement stepCycle: LAST / Resolution: 1.84→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5728 0 49 605 6382
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.0226050
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.6621.9398217
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.8995747
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.75423.618293
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.85115988
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.8661543
X-RAY DIFFRACTIONr_chiral_restr0.1320.2860
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.024740
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined0.2140.23137
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined0.3140.24237
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1540.2562
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1740.2198
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1630.250
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.9871.53685
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.6725958
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it3.02732404
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it4.7074.52259
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.84→1.89 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.302 101
Rwork0.266 4929

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