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- PDB-5oft: Structural basis for OXA-48 dimerization -

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Basic information

Entry
Database: PDB / ID: 5oft
TitleStructural basis for OXA-48 dimerization
ComponentsBeta-lactamase
KeywordsANTIBIOTIC / dimerization / OXA-48 / Oxacillinase / carbapenemase / beta-lactamase
Function / homology
Function and homology information


penicillin binding / antibiotic catabolic process / cell wall organization / beta-lactamase activity / beta-lactamase / response to antibiotic / metal ion binding
Similarity search - Function
Beta-lactamase, class-D active site / Beta-lactamase class-D active site. / Penicillin-binding protein, transpeptidase / Penicillin binding protein transpeptidase domain / Beta-lactamase / DD-peptidase/beta-lactamase superfamily / Beta-lactamase/transpeptidase-like / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesKlebsiella pneumoniae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.2 Å
AuthorsLund, B.A. / Nesheim, B.H.B. / Leiros, H.K.S.
CitationJournal: FEBS J / Year: 2018
Title: The biological assembly of OXA-48 reveals a dimer interface with high charge complementarity and very high affinity.
Authors: Bjarte Aarmo Lund / Ane Molden Thomassen / Birgit Helene Berg Nesheim / Trine Josefine Olsen Carlsen / Johan Isaksson / Tony Christopeit / Hanna-Kirsti S Leiros /
Abstract: Many class D β-lactamases form dimers in solution. The functional basis of the dimerization of OXA-48-like class D β-lactamases is not known, but in order to understand the structural requirements ...Many class D β-lactamases form dimers in solution. The functional basis of the dimerization of OXA-48-like class D β-lactamases is not known, but in order to understand the structural requirements for dimerization of OXA-48, we have characterized the dimer interface. Size exclusion chromatography, small angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) were used to confirm the oligomeric state of OXA-48 in solution. X-ray crystallographic structures were used to elucidate the key interactions of dimerization. In silico residue scanning combined with site-directed mutagenesis was used to probe hot spots of dimerization. The affinity of dimerization was quantified using microscale thermophoresis, and the overall thermostability was investigated using differential scanning calorimetry. OXA-48 was consistently found to be a dimer in solution regardless of the method used, and the biological assembly found from the SAXS envelope is consistent with the dimer identified from the crystal structures. The buried chloride that interacts with Arg206 and Arg206' at the dimer interface was found to enhance the thermal stability by > 4 °C and crystal structures and mutations (R189A, R189A/R206A) identified several additional important ionic interactions. The affinity for OXA-48 R206A dimerization was in the picomolar range, thus revealing very high dimer affinity. In summary, OXA-48 has a very stable dimer interface, facilitated by noncovalent and predominantly charged interactions, which is stronger than the dimer interfaces previously described for other class D β-lactamases. PDB CODES: The oxacillinase-48 (OXA-48) R206A structure has PDB ID: 5OFT and OXA-48 R189A has PDB ID: 6GOA.
History
DepositionJul 11, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 1, 2018Provider: repository / Type: Initial release
Revision 1.1Aug 29, 2018Group: Data collection / Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Sep 5, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.3Nov 28, 2018Group: Data collection / Database references / Category: citation / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Jan 17, 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: Beta-lactamase
B: Beta-lactamase


Theoretical massNumber of molelcules
Total (without water)56,2822
Polymers56,2822
Non-polymers00
Water79344
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1810 Å2
ΔGint3 kcal/mol
Surface area20050 Å2
MethodPISA
Unit cell
Length a, b, c (Å)120.151, 120.151, 160.107
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number179
Space group name H-MP6522

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Components

#1: Protein Beta-lactamase


Mass: 28140.836 Da / Num. of mol.: 2 / Mutation: R206A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: bla OXA-48, blaOXA-48, KPE71T_00045 / Plasmid: pDEST17 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Star pRARE / References: UniProt: Q6XEC0, beta-lactamase
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 44 / 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: 2.96 Å3/Da / Density % sol: 58.5 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 9
Details: 14-18% PEG MME 5K and 0.1 M Bis Tris Propane pH 9-9.5. 2 uL hanging drops with a 1:1 ratio between protein and reservoir solution were equilibrated over 1 mL of reservoir solution
PH range: 9-9.5 / Temp details: room temperature

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.9184 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jan 13, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 3.2→35.3 Å / Num. obs: 11089 / % possible obs: 93.74 % / Redundancy: 3.3 % / Rrim(I) all: 0.185 / Net I/σ(I): 8.36
Reflection shellResolution: 3.2→3.314 Å / Redundancy: 3.4 % / Mean I/σ(I) obs: 2.45 / Num. measured obs: 3732 / Num. unique all: 1101 / % possible all: 96.33

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5dtk

5dtk


Resolution: 3.2→35.299 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 23.09
RfactorNum. reflection% reflection
Rfree0.2425 548 4.94 %
Rwork0.1824 --
obs0.1855 11084 93.87 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.2→35.299 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3946 0 0 44 3990
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0064044
X-RAY DIFFRACTIONf_angle_d0.6755478
X-RAY DIFFRACTIONf_dihedral_angle_d18.2482372
X-RAY DIFFRACTIONf_chiral_restr0.045578
X-RAY DIFFRACTIONf_plane_restr0.005706
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2001-3.52190.32371380.24692592X-RAY DIFFRACTION95
3.5219-4.03090.20931340.19232598X-RAY DIFFRACTION95
4.0309-5.07610.22921340.15062623X-RAY DIFFRACTION94
5.0761-35.3010.23271420.16882723X-RAY DIFFRACTION92
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)
10.54220.0139-0.49092.2053-1.18851.0762-0.5562-0.0897-0.60550.1710.05680.0999-0.14520.16270.13460.9410.1550.11550.2072-0.02870.8881-49.1094-7.6291-22.5789
21.77870.4078-0.07532.7556-0.39621.6631-0.3138-0.038-0.1151-0.14830.0305-0.26820.67590.05680.20670.8878-0.020.12550.1985-0.12780.5134-51.2482-1.0924-25.6597
32.03880.5141-0.14693.3133-0.42561.0407-0.1986-0.0204-0.2286-0.03330.26440.850.4321-0.1949-0.02570.5183-0.06080.09620.23210.02540.2918-59.61529.0264-17.4861
43.42430.19330.2580.9706-0.41612.92290.1604-0.30040.26220.56850.43380.0892-0.4719-0.07-0.62490.5214-0.0006-0.06310.2546-0.07860.4673-53.85828.3131-8.9386
51.7561-0.6242-1.52842.8084-0.84042.07240.2424-0.0385-0.53030.3155-0.2392-0.2266-0.89550.47630.04070.4825-0.0614-0.09390.26980.0990.403-48.536922.8969-9.5617
63.22470.49750.82792.2246-0.08115.0956-0.2071-0.10710.7961-0.2479-0.08190.3405-1.3019-0.9590.2110.50470.1415-0.07030.4235-0.01790.4105-66.028121.7841-13.6773
72.4783-1.1616-0.89155.0627-2.39422.068-0.60080.0079-0.0953-0.14680.29831.0063-0.2502-0.8176-0.11120.41110.0137-0.05630.44740.10940.3032-68.345813.1677-14.5594
82.0906-0.9597-1.11022.1558-0.17613.1622-0.26250.31520.0612-0.3220.0030.08180.2691-0.45960.28880.4813-0.034-0.07590.31680.01130.2718-59.669214.801-23.6499
91.81750.37610.70441.28930.29211.169-0.13430.0341-0.1349-0.1282-0.0381-0.150.50060.01880.12780.53730.03770.05840.23830.04240.3807-47.13086.0132-19.3205
103.1787-0.349-0.97030.7979-1.41864.11080.5711-0.4477-0.31490.719-0.2585-0.9552-0.84040.9361-0.19220.6852-0.3799-0.3140.86810.16680.7553-19.883535.1086-22.2028
114.29710.0639-1.24144.00240.68731.8298-0.3144-0.05740.73920.5879-0.0623-0.2896-0.57360.80810.25490.5867-0.2244-0.18440.60860.15370.4762-27.039937.8234-24.2823
123.3438-0.53880.0181.3343-0.16723.0509-0.07290.9148-0.24920.3185-0.0615-0.261-0.14090.35210.20070.33350.0358-0.07160.3670.0330.3389-42.228.9798-41.4511
133.1311-0.00210.41432.48710.46662.71220.30370.3935-0.48840.0816-0.1411-0.08270.73370.2898-0.2240.53020.007-0.01220.4608-0.05160.3814-37.379516.2925-46.245
146.859-3.9433-0.37417.3427-1.5010.59940.64540.817-0.6312-0.9484-0.31180.0280.7958-0.1724-0.26630.4294-0.0338-0.0080.2866-0.08650.2827-41.487426.5732-49.8069
157.494-2.93891.98384.97242.68813.67580.10250.41070.6228-0.04180.2193-0.5250.012-0.78720.36360.6289-0.11790.00630.39630.0630.1947-44.866640.2365-46.412
161.6160.64950.0571.5922-0.3570.2957-0.0787-0.06550.18560.2813-0.2063-0.211-0.11880.16060.16650.3925-0.1283-0.05280.38240.02890.4105-35.922737.679-40.0737
173.26780.8636-0.47631.9108-1.86286.3981-0.0220.18060.10530.24070.56130.084-0.7666-0.6374-0.28220.4057-0.087-0.09550.37550.05090.2743-48.956431.6985-32.2777
182.01050.96540.67852.7314-0.29150.98740.105-0.2072-0.51070.1979-0.2175-0.5249-0.26730.93060.17530.3818-0.083-0.10370.51240.10650.4075-29.107326.806-28.4698
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 24 through 37 )
2X-RAY DIFFRACTION2chain 'A' and (resid 38 through 56 )
3X-RAY DIFFRACTION3chain 'A' and (resid 57 through 82 )
4X-RAY DIFFRACTION4chain 'A' and (resid 83 through 102 )
5X-RAY DIFFRACTION5chain 'A' and (resid 103 through 119 )
6X-RAY DIFFRACTION6chain 'A' and (resid 120 through 141 )
7X-RAY DIFFRACTION7chain 'A' and (resid 142 through 155 )
8X-RAY DIFFRACTION8chain 'A' and (resid 156 through 194 )
9X-RAY DIFFRACTION9chain 'A' and (resid 195 through 265 )
10X-RAY DIFFRACTION10chain 'B' and (resid 24 through 38 )
11X-RAY DIFFRACTION11chain 'B' and (resid 39 through 63 )
12X-RAY DIFFRACTION12chain 'B' and (resid 64 through 92 )
13X-RAY DIFFRACTION13chain 'B' and (resid 93 through 119 )
14X-RAY DIFFRACTION14chain 'B' and (resid 120 through 130 )
15X-RAY DIFFRACTION15chain 'B' and (resid 131 through 141 )
16X-RAY DIFFRACTION16chain 'B' and (resid 142 through 177 )
17X-RAY DIFFRACTION17chain 'B' and (resid 178 through 194 )
18X-RAY DIFFRACTION18chain 'B' and (resid 195 through 265 )

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