SASDEM3: Dimeric class D beta-lactamase OXA-48 (Beta-lactamase, OXA-48)

Basic information

• Entry: Database: SASBDB / ID: SASDEM3

Sample

Dimeric class D beta-lactamase OXA-48

• Beta-lactamase (protein), OXA-48, Klebsiella pneumoniae

Function / homology

Function:

penicillin binding / antibiotic catabolic process / beta-lactamase activity / beta-lactamase / response to antibiotic / metal ion binding

Domain/homology:

Beta-lactamase, class-D active site / Beta-lactamase class-D active site. / : / Penicillin-binding protein, transpeptidase / Penicillin binding protein transpeptidase domain / Beta-lactamase/transpeptidase-like

Component:

Beta-lactamase

• Biological species: Klebsiella pneumoniae (bacteria)
• Citation: Journal: 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 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.

Contact author

• Bjarte Aarmo Lund (UiT, UiT - The Arctic University of Norway, Tromso, Norway)

Models

• Model #1389: ; Type: atomic / Radius of dummy atoms: 1.90 A / Comment: chains A and D / Chi-square value: 5.025; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Dimeric class D beta-lactamase OXA-48 / Specimen concentration: 0.10-13.00
• Buffer: Name: 50 mM HEPES 50 mM K2SO4 / pH: 7

Entity #741

Name: OXA-48 / Type: protein / Description: Beta-lactamase / Formula weight: 28.218 / Num. of mol.: 2 / Source: Klebsiella pneumoniae / References: UniProt: Q6XEC0
Sequence:

AKEWQENKSW NAHFTEHKSQ GVVVLWNENK QQGFTNNLKR ANQAFLPAST FKIPNSLIAL DLGVVKDEHQ VFKWDGQTRD IATWNRDHNL ITAMKYSVVP VYQEFARQIG EARMSKMLHA FDYGNEDISG NVDSFWLDGG IRISATEQIS FLRKLYHNKL HVSERSQRIV KQAMLTEANG DYIIRAKTGY STRIEPKIGW WVGWVELDDN VWFFAMNMDM PTSDGLGLRQ AITKEVLKQE KIIP

Experimental information

• Beam: Instrument name: ESRF BM29 / City: Grenoble / 国: France / Type of source: X-ray synchrotron / Wavelength: 0.09919 Å / Dist. spec. to detc.: 2.867 mm
• Detector: Name: Pilatus 1M / Type: Dectris / Pixsize x: 172 mm

Scan

Title: Dimeric class D beta-lactamase OXA-48 / Measurement date: Feb 23, 2017 / Storage temperature: 25 °C / Cell temperature: 25 °C / Exposure time: 10 sec. / Number of frames: 10 / Unit: 1/nm /

	Min	Max
Q	0.0788	4.8993

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 671 /

	Min	Max
Q	0.0835416	3.23758
P(R) point	1	671
R	0	7.35

Result

Type of curve: merged /

	Experimental	Porod
MW	50 kDa	46 kDa
Volume	-	74 nm3

	P(R)	Guinier	Guinier error
Forward scattering, I0	26.79	27.04	0.052
Radius of gyration, Rg	2.476 nm	2.47 nm	0.018

	Min	Max
D	-	7.35
Guinier point	2	96