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- PDB-1ia3: Candida albicans dihydrofolate reductase complex in which the dih... -

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Basic information

Entry
Database: PDB / ID: 1ia3
TitleCandida albicans dihydrofolate reductase complex in which the dihydronicotinamide moiety of dihydro-nicotinamide-adenine-dinucleotide phosphate (NADPH) is displaced by 5-[(4-TERT-BUTYLPHENYL)SULFANYL]-2,4-QUINAZOLINEDIAMINE (GW995)
ComponentsDIHYDROFOLATE REDUCTASE
KeywordsOXIDOREDUCTASE / ANTIFUNGAL TARGET / REDUCTASE / Dihydronicotinamide displaced
Function / homology
Function and homology information


dihydrofolate metabolic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / tetrahydrofolate biosynthetic process / one-carbon metabolic process / NADP binding / mitochondrion
Similarity search - Function
Dihydrofolate reductase / Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase-like domain superfamily / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-NDP / Chem-TQ5 / Dihydrofolate reductase
Similarity search - Component
Biological speciesCandida albicans (yeast)
MethodX-RAY DIFFRACTION / DIRECT REPLACEMENT / Resolution: 1.78 Å
AuthorsWhitlow, M. / Howard, A.J. / Kuyper, L.F.
Citation
Journal: J.Med.Chem. / Year: 2001
Title: X-ray Crystal Structures of Candida albicans Dihydrofolate Reductase: High Resolution Ternary Complexes in Which the Dihydronicotinamide Moiety of NADPH is Displaced by an inhibitor
Authors: Whitlow, M. / Howard, A.J. / Stewart, D. / Hardman, K.D. / Chan, J.H. / Baccanari, D.P. / Tansik, R.L. / Hong, J.S. / Kuyper, L.F.
#1: Journal: J.Biol.Chem. / Year: 1997
Title: X-ray Crystallographic Studies of Candida albicans Dihydrofolate Reductase. High resolution structures of the holoenzyme and an inhibited ternary complex.
Authors: Whitlow, M. / Howard, A.J. / Stewart, D. / Hardman, K.D. / Kuyper, L.F. / Baccanari, D.P. / Fling, M.E. / Tansik, R.L.
#2: Journal: J.Med.Chem. / Year: 1995
Title: Selective Inhibitors of Candida albicans Dihydrofolate Reductase: Activity and Selectivity of 5-(arylthio)-2,4-diaminoquinazolines
Authors: Chan, J.H. / Hong, J.S. / Kuyper, L.F. / Baccanari, D.P. / Joyner, S.S. / Tansik, R.L. / Boytos, C.M. / Rudolph, S.K.
#3: Journal: J.Biol.Chem. / Year: 1989
Title: Characterization of Candida Albicans Dihydrofolate Reductase
Authors: Baccanari, D.P. / Tansik, R.L. / Joyner, S.S. / Fling, M.E. / Smith, P.L. / Freisheim, J.H.
History
DepositionMar 22, 2001Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 11, 2001Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Oct 4, 2017Group: Refinement description / Category: software / Item: _software.name
Revision 1.4Aug 9, 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 / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 99HET GROUPS NDP 193, TQ5 194, AND MES 201 ARE ASSOCIATED WITH PROTEIN CHAIN A. HET GROUPS NDP 195, ...HET GROUPS NDP 193, TQ5 194, AND MES 201 ARE ASSOCIATED WITH PROTEIN CHAIN A. HET GROUPS NDP 195, TQ5 196, AND MES 202 ARE ASSOCIATED WITH PROTEIN CHAIN B.
Remark 600 HETEROGEN THE INHIBITION CONSTANT (IC50) FOR 5-[(4-TERT-BUTYLPHENYL) SULFANYL]-2,4- ... HETEROGEN THE INHIBITION CONSTANT (IC50) FOR 5-[(4-TERT-BUTYLPHENYL) SULFANYL]-2,4-QUINAZOLINEDIAMINE (GW995) IS 8 NANOMOLAR USING THE FOLLOWING ASSAY (SEE REFERENCE 0 & 3). C. ALBICANS DHFR ASSAY WAS PERFORMED IN 0.1 M IMIDAZOLE CHLORIDE BUFFER, PH 6.4, WITH 12 MM MERCAPTOETHANOL, 60 MM NADPH AND 45 MM DIHYDROFOLIC ACID IN A FINAL VOLUME OF 1 ML AT 303K. IC50 IS THE CONCENTRATION OF INHIBITOR THAT DECREASES THE VELOCITY OF THE STANDARD ASSAY BY 50%. THE ENZYME (0.2 NM), NADPH, AND VARYING CONCENTRATIONS OF INHIBITOR WERE PREINCUBATED FOR 2 MIN AT 30-C, AND THE REACTION WAS INITIATED BY THE ADDITION OF DIHYDROFOLIC ACID. STEADY STATE VELOCITIES WERE MEASURED, AND IC50 VALUES WERE CALCULATED FROM A LINEAR REGRESSION PLOT OF THE PERCENTAGE INHIBITION VS THE LOGARITHM OF THE INHIBITOR CONCENTRATION. IC50 THE PRECISION OF THE DETERMINATION IS GENERALLY ABOUT A 30%.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DIHYDROFOLATE REDUCTASE
B: DIHYDROFOLATE REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,9198
Polymers44,3892
Non-polymers2,5306
Water6,107339
1
A: DIHYDROFOLATE REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,4604
Polymers22,1951
Non-polymers1,2653
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: DIHYDROFOLATE REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,4604
Polymers22,1951
Non-polymers1,2653
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)76.91, 67.28, 38.49
Angle α, β, γ (deg.)90.00, 93.07, 90.00
Int Tables number4
Space group name H-MP1211
DetailsDihydrofolate reductase is active as a monomer.

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Components

#1: Protein DIHYDROFOLATE REDUCTASE


Mass: 22194.527 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candida albicans (yeast) / Gene: DFR1 / Plasmid: P1869 / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P22906, dihydrofolate reductase
#2: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#3: Chemical ChemComp-TQ5 / 5-[4-TERT-BUTYLPHENYLSULFANYL]-2,4-QUINAZOLINEDIAMINE


Mass: 324.443 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H20N4S
#4: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID


Mass: 195.237 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 339 / 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: 2.27 Å3/Da / Density % sol: 43 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: DIHYDRO-NICOTINAMIDE-ADENINE- DINUCLEOTIDE PHOSPHATE (NADPH),5-[(4-TERT-BUTYLPHENYL) SULFANYL]-2,4-QUINAZOLINEDIAMINE (GW995), PEG-3350, POTASSIUM 4-MORPHILINEERHANESULFONIC ACID, ...Details: DIHYDRO-NICOTINAMIDE-ADENINE- DINUCLEOTIDE PHOSPHATE (NADPH),5-[(4-TERT-BUTYLPHENYL) SULFANYL]-2,4-QUINAZOLINEDIAMINE (GW995), PEG-3350, POTASSIUM 4-MORPHILINEERHANESULFONIC ACID, DITHIOTHREITOL A THREE-FOLD EXCESS OF GW995 AND THREE-FOLD EXCESS OF NADPH WAS ADDED TO THE C. ALBICANS DHFR SOLUTION AND LET STAND 277K OVERNIGHT. 17-20 MG/ML C. ALBICANS DHFR IN 50 UM GW995, 50 UM NADPH, 20 MM KMES, 1 MM DTT, PH 6.5 WAS MIXED WITH AN EQUAL PART OF 26 - 34% PEG-3350, THE RESERVOIR SOLUTION. , VAPOR DIFFUSION, HANGING DROP
Crystal grow
*PLUS
Temperature: 4 ℃
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
117-20 mg/mlprotein1drop
250 mMKMES1drop
31 mMNADPH1drop
41 mMdithiothreitol1drop
526-34 %PEG33501reservoir

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Data collection

DiffractionMean temperature: 293 K
Diffraction sourceSource: ROTATING ANODE / Type: ELLIOTT GX-21 / Wavelength: 1.5418
DetectorType: XENTRONICS / Detector: AREA DETECTOR / Date: Aug 30, 1988 / Details: Huber graphite monochromator
RadiationMonochromator: Huber graphite monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.75→25 Å / Num. all: 116944 / Num. obs: 36058 / % possible obs: 90.1 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 3.24 % / Biso Wilson estimate: 36.3 Å2 / Rmerge(I) obs: 0.0549 / Rsym value: 0.0549 / Net I/σ(I): 16.18
Reflection shellResolution: 1.75→1.85 Å / Redundancy: 1.11 % / Rmerge(I) obs: 0.2694 / Mean I/σ(I) obs: 2.23 / Num. unique all: 4005 / Rsym value: 0.2694 / % possible all: 60.4
Reflection shell
*PLUS
% possible obs: 60.4 %

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Processing

Software
NameClassification
X-GENdata reduction
FRODOmodel building
PROFFTrefinement
X-GENdata scaling
RefinementMethod to determine structure: DIRECT REPLACEMENT
Starting model: CANDIDA ALBICANS DHFR (PDB ENTRY 1AI9)

1ai9


Resolution: 1.78→10 Å
Isotropic thermal model: Konnert, J.H. & Hendrickson, W.A. (1980) Acta Crystallogr. A A36, 344.
σ(F): 2 / σ(I): 0
Stereochemistry target values: Hendrickson, W.A. (1985) Methods Enzymol. 115, 252-270.
Details: Restrain least-squares procedure
RfactorNum. reflection% reflection
Rwork0.16 --
all0.16 36058 -
obs0.16 32308 84.6 %
Refinement stepCycle: LAST / Resolution: 1.78→10 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3181 0 140 350 3671
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONp_bond_d0.0210.03
X-RAY DIFFRACTIONp_angle_d0.0340.04
X-RAY DIFFRACTIONp_planar_d0.040.05
X-RAY DIFFRACTIONp_hb_or_metal_coord0.1
X-RAY DIFFRACTIONp_mcbond_it0.8471
X-RAY DIFFRACTIONp_mcangle_it1.4622
X-RAY DIFFRACTIONp_scbond_it2.4012.5
X-RAY DIFFRACTIONp_scangle_it3.8265
X-RAY DIFFRACTIONp_plane_restr0.0210.03
X-RAY DIFFRACTIONp_chiral_restr0.2430.3
X-RAY DIFFRACTIONp_singtor_nbd0.1670.2
X-RAY DIFFRACTIONp_multtor_nbd0.1480.2
X-RAY DIFFRACTIONp_xhyhbond_nbd0.2
X-RAY DIFFRACTIONp_xyhbond_nbd0.1360.2
X-RAY DIFFRACTIONp_planar_tor3.86
X-RAY DIFFRACTIONp_staggered_tor14.910
X-RAY DIFFRACTIONp_transverse_tor32.610
X-RAY DIFFRACTIONp_special_tor6
LS refinement shell
Resolution (Å)Rfactor RworkRefine-IDNum. reflection obs
1.78-20.192X-RAY DIFFRACTION7123
2-2.160.17X-RAY DIFFRACTION4897
2.16-2.390.161X-RAY DIFFRACTION5136
2.39-2.770.163X-RAY DIFFRACTION5394
2.77-3.630.143X-RAY DIFFRACTION5470
3.63-100.153X-RAY DIFFRACTION4288

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