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- PDB-2xdf: Solution Structure of the Enzyme I Dimer Complexed with HPr Using... -

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Basic information

Entry
Database: PDB / ID: 2xdf
TitleSolution Structure of the Enzyme I Dimer Complexed with HPr Using Residual Dipolar Couplings and Small Angle X-Ray Scattering
Components
  • PHOSPHOCARRIER PROTEIN HPR
  • PHOSPHOENOLPYRUVATE-PROTEIN PHOSPHOTRANSFERASE
KeywordsTRANSFERASE / SUGAR TRANSPORT
Function / homology
Function and homology information


phosphotransferase activity, nitrogenous group as acceptor / phosphoenolpyruvate-protein phosphotransferase / phosphoenolpyruvate-protein phosphotransferase activity / N-acetylglucosamine transport / antisigma factor binding / positive regulation of glycogen catabolic process / regulation of carbon utilization / phosphoenolpyruvate-dependent sugar phosphotransferase system / enzyme inhibitor activity / enzyme regulator activity ...phosphotransferase activity, nitrogenous group as acceptor / phosphoenolpyruvate-protein phosphotransferase / phosphoenolpyruvate-protein phosphotransferase activity / N-acetylglucosamine transport / antisigma factor binding / positive regulation of glycogen catabolic process / regulation of carbon utilization / phosphoenolpyruvate-dependent sugar phosphotransferase system / enzyme inhibitor activity / enzyme regulator activity / enzyme activator activity / kinase activity / metal ion binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Phosphotransferase system, enzyme I / Phosphotransferase system, enzyme I-like / Phosphotransferase system, enzyme I N-terminal / PtsI, HPr-binding domain superfamily / : / PEP-utilising enzyme, N-terminal / PEP-utilising enzyme, active site / PEP-utilizing enzymes phosphorylation site signature. / PEP-utilising enzyme, conserved site / PEP-utilizing enzymes signature 2. ...Phosphotransferase system, enzyme I / Phosphotransferase system, enzyme I-like / Phosphotransferase system, enzyme I N-terminal / PtsI, HPr-binding domain superfamily / : / PEP-utilising enzyme, N-terminal / PEP-utilising enzyme, active site / PEP-utilizing enzymes phosphorylation site signature. / PEP-utilising enzyme, conserved site / PEP-utilizing enzymes signature 2. / PEP-utilising enzyme, C-terminal / PEP-utilising enzyme, PEP-binding domain / Phosphotransferase system, HPr histidine phosphorylation site / PEP-utilising enzyme, mobile domain / Phosphohistidine domain superfamily / PEP-utilising enzyme, mobile domain / PTS HPR domain histidine phosphorylation site signature. / Phosphotransferase system, HPr serine phosphorylation site / HPr-like / Phosphocarrier protein HPr-like / HPr-like superfamily / : / PTS HPr component phosphorylation site / PTS HPR domain profile. / Histidine-containing Protein; Chain: A; / PTS HPR domain serine phosphorylation site signature. / Phosphoenolpyruvate-binding domains / Pyruvate kinase-like domain superfamily / Pyruvate/Phosphoenolpyruvate kinase-like domain superfamily / TIM Barrel / Alpha-Beta Barrel / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Phosphoenolpyruvate-protein phosphotransferase / Phosphocarrier protein HPr / Phosphocarrier protein HPr
Similarity search - Component
Biological speciesESCHERICHIA COLI (E. coli)
MethodSOLUTION NMR / SOLUTION SCATTERING / simulated annealing
AuthorsSchwieters, C.D. / Suh, J.-Y. / Grishaev, A. / Guirlando, R. / Takayama, Y. / Clore, G.M.
CitationJournal: J Am Chem Soc / Year: 2010
Title: Solution structure of the 128 kDa enzyme I dimer from Escherichia coli and its 146 kDa complex with HPr using residual dipolar couplings and small- and wide-angle X-ray scattering.
Authors: Charles D Schwieters / Jeong-Yong Suh / Alexander Grishaev / Rodolfo Ghirlando / Yuki Takayama / G Marius Clore /
Abstract: The solution structures of free Enzyme I (EI, ∼128 kDa, 575 × 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr (∼146 kDa) have been solved using ...The solution structures of free Enzyme I (EI, ∼128 kDa, 575 × 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr (∼146 kDa) have been solved using novel methodology that makes use of prior structural knowledge (namely, the structures of the dimeric EIC domain and the isolated EIN domain both free and complexed to HPr), combined with residual dipolar coupling (RDC), small- (SAXS) and wide- (WAXS) angle X-ray scattering and small-angle neutron scattering (SANS) data. The calculational strategy employs conjoined rigid body/torsion/Cartesian simulated annealing, and incorporates improvements in calculating and refining against SAXS/WAXS data that take into account complex molecular shapes in the description of the solvent layer resulting in a better representation of the SAXS/WAXS data. The RDC data orient the symmetrically related EIN domains relative to the C(2) symmetry axis of the EIC dimer, while translational, shape, and size information is provided by SAXS/WAXS. The resulting structures are independently validated by SANS. Comparison of the structures of the free EI and the EI-HPr complex with that of the crystal structure of a trapped phosphorylated EI intermediate reveals large (∼70-90°) hinge body rotations of the two subdomains comprising the EIN domain, as well as of the EIN domain relative to the dimeric EIC domain. These large-scale interdomain motions shed light on the structural transitions that accompany the catalytic cycle of EI.
History
DepositionApr 30, 2010Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 22, 2010Provider: repository / Type: Initial release
Revision 1.1Dec 21, 2011Group: Database references / Version format compliance
Revision 1.2Mar 22, 2017Group: Data collection
Revision 1.3Aug 21, 2019Group: Data collection / Category: pdbx_nmr_software / Item: _pdbx_nmr_software.name
Revision 1.4May 15, 2024Group: Data collection / Database references / Other
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_mr
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AB" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AB" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 9-STRANDED BARREL THIS IS REPRESENTED BY A 10-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "BB" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 9-STRANDED BARREL THIS IS REPRESENTED BY A 10-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: PHOSPHOENOLPYRUVATE-PROTEIN PHOSPHOTRANSFERASE
B: PHOSPHOENOLPYRUVATE-PROTEIN PHOSPHOTRANSFERASE
C: PHOSPHOCARRIER PROTEIN HPR
D: PHOSPHOCARRIER PROTEIN HPR


Theoretical massNumber of molelcules
Total (without water)145,0974
Polymers145,0974
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)2 / 120BEST EXPERIMENT FIT, AND THEN LOWEST ENERGY
RepresentativeModel #1

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Components

#1: Protein PHOSPHOENOLPYRUVATE-PROTEIN PHOSPHOTRANSFERASE / PHOSPHOTRANSFERASE SYSTEM ENZYME I


Mass: 63419.344 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Strain: BL21 STAR (DE3) / Plasmid: PET11 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 STAR (DE3)
References: UniProt: P08839, phosphoenolpyruvate-protein phosphotransferase
#2: Protein PHOSPHOCARRIER PROTEIN HPR / HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR


Mass: 9129.332 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Strain: BL21 STAR (DE3) / Plasmid: PET11 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 STAR (DE3)
References: UniProt: P0AA06, UniProt: P0AA04*PLUS, Transferases; Transferring phosphorus-containing groups; Protein-serine/threonine kinases

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Experimental details

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Experiment

Experiment
Method
SOLUTION NMR
SOLUTION SCATTERING
NMR experimentType: TROSY-BASED 1H-15N CORRELATION SPECTROSCOPY

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Sample preparation

DetailsContents: 90% H2O/10% D2O
Sample conditionspH: 7.4 / Pressure: 1.0 atm / Temperature: 310.0 K

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Data collection

NMR spectrometerType: Bruker DRX / Manufacturer: Bruker / Model: DRX / Field strength: 800 MHz
Soln scatter

Data analysis software list: GNOM / Protein length: 16 / Temperature: 298 K

TypeIDConc. range (mg/ml)Detector typeMean guiner radius (nm)Num. of time framesSource beamlineSource classSource type
x-ray12.5-5GOLD CCD4.59202.12-IDCsynchrotronALS
neutron254.69neutron sourceNIST 30M NG3

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Processing

NMR software
NameVersionClassification
Xplor-NIH2.25refinement
Xplor-NIHstructure solution
RefinementMethod: simulated annealing / Software ordinal: 1
Details: REFINEMENT DETAILS CAN BE FOUND IN THE PRIMARY CITATION. THE INITIAL STRUCTURE OF THE EI DIMER COMPLEXED WITH HPR WAS CONSTRUCTED AS A HYBRID OF THE CRYSTAL STRUCTURE OF PHOSPHORYLATED EI ...Details: REFINEMENT DETAILS CAN BE FOUND IN THE PRIMARY CITATION. THE INITIAL STRUCTURE OF THE EI DIMER COMPLEXED WITH HPR WAS CONSTRUCTED AS A HYBRID OF THE CRYSTAL STRUCTURE OF PHOSPHORYLATED EI INTERMEDIATE CAPTURED BY THE INHIBITOR OXALATE (PDB CODE 2HWG) AND THE NMR STRUCTURE OF THE EIN-HPR COMPLEX (PDB CODE 3EZA). THROUGHOUT THE STRUCTURE DETERMINATION, THE BACKBONE ATOMIC COORDINATES OF EACH EIN-HPR COMPLEX (RESIDUES 1-254 AND 601-685) WERE TREATED AS RIGID BODIES, WITH THE TWO SYMMETRY RELATED EIC DOMAINS (RESIDUES 262- 573) HELD FIXED IN SPACE. COORDINATES IN THE LINKER REGION (RESIDUES 255-261) WERE ALLOWED VARYING DEGREES OF FREEDOM DURING THE CALCULATION THROUGH THE USE OF THE INTERNAL VARIABLE MODULE (IVM) OF XPLOR-NIH. THE ENSEMBLE OF CALCULATED STRUCTURES FELL IN TWO CLUSTERS, THE REGULARIZED REFINED MEAN OF EACH IS INCLUDED BELOW AS MODELS 1 AND 2, RESPECTIVELY. STRUCTURAL STATISTICS: CLUSTER 1: SAXS CHI2 Q->0.44 0.63+/-0.11 SAXS CHI2 FULL RANGE FIT 0.45+/-0.07 SANS CHI2 1.38+/-0.51 RDC R-FACTOR 16.30+/-0.03 % RDC DA 13.73+/-0.05 HZ RDC RHOMBICITY 0.63+/-0.00 MODEL 1: SAXS CHI2 Q->0.44 0.62 SAXS CHI2 FULL RANGE FIT 0.42 SANS CHI2 1.30 RDC R-FACTOR 16.27 % RDC DA 13.74 HZ RDC RHOMBICITY 0.63 STRUCTURAL STATISTICS: CLUSTER 2 SAXS CHI2 Q->0.44 0.76+/-0.07 SAXS CHI2 FULL RANGE FIT 0.48+/-0.06 SANS CHI2 2.97+/-0.62 RDC R-FACTOR 16.25+/-0.02 % RDC DA 13.83+/-0.08 HZ RDC RHOMBICITY 0.63+/-0.00 MODEL 2 SAXS CHI2 Q->0.44 0.78 SAXS CHI2 FULL RANGE FIT 0.49 SANS CHI2 2.82 RDC R-FACTOR 16.25 % RDC DA 13.85 HZ RDC RHOMBICITY 0.63
NMR ensembleConformer selection criteria: BEST EXPERIMENT FIT, AND THEN LOWEST ENERGY
Conformers calculated total number: 120 / Conformers submitted total number: 2

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