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Studying the active-site loop movement of the São Paolo metallo-β-lactamase-1†Electronic supplementary information (ESI) available: Procedures for protein expression and purification, (19)F-labelling, crystallisation, data collection, and structure determination, table of crystallographic data, table of crystallographic parameters and refinement statistics, figures showing binding mode and distances, procedures for mass spectrometry measurements, differential scanning fluorimetry measurements, stopped-flow measurements and other kinetics measurements. See DOI: 10.1039/c4sc01752hClick here for additional data file.

Brem J, Struwe WB, Rydzik AM, Tarhonskaya H, Pfeffer I, Flashman E, van Berkel SS, Spencer J, Claridge TD, McDonough MA, Benesch JL, Schofield CJ - Chem Sci (2014)

Bottom Line: These include a mobile α3 region which is important in catalysis and determining inhibitor selectivity.SPM-1 thus appears to be a hybrid B1/B2 MBL.The results have implications for MBL evolution and inhibitor design.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry , University of Oxford , 12 Mansfield Road , Oxford , OX1 3TA , UK . Email: christopher.schofield@chem.ox.ac.uk.

ABSTRACT

Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibiotics. We report biophysical and kinetic studies on the São Paulo MBL (SPM-1), which reveal its Zn(ii) ion usage and mechanism as characteristic of the clinically important di-Zn(ii) dependent B1 MBL subfamily. Biophysical analyses employing crystallography, dynamic (19)F NMR and ion mobility mass spectrometry, however, reveal that SPM-1 possesses loop and mobile element regions characteristic of the B2 MBLs. These include a mobile α3 region which is important in catalysis and determining inhibitor selectivity. SPM-1 thus appears to be a hybrid B1/B2 MBL. The results have implications for MBL evolution and inhibitor design.

No MeSH data available.


Non-denaturing ion mobility MS reveals different conformations of di-Zn(ii)-SPM-1 as compared to the theoretical collisional cross section (CCS) values of the ‘open’ and ‘closed’ crystal forms.
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fig3: Non-denaturing ion mobility MS reveals different conformations of di-Zn(ii)-SPM-1 as compared to the theoretical collisional cross section (CCS) values of the ‘open’ and ‘closed’ crystal forms.

Mentions: IM-MS was used to investigate different conformational states of SPM-1 with and without Zn bound through reporting their corresponding CCS values. An IM-MS spectrum under conditions that preserve non-covalent interaction shows three major charge states (9+ → 11+) of SPM-1 with distributions of z = 9+ (10%), 10+ (60%), 11+ (30%), corresponding to a mass of 28 030.5 Da (Fig. 2A). The CCS of the charge states are: 2119 Å2 (9+), 2167 Å2 (10+) and 2247 Å2 (11+) (Fig. 3B). The calculated CCS from X-ray structures of ‘open’ and ‘closed’ forms of SPM-1 are 2237 Å2 (open) and 2171 Å2 (closed). The ‘open’ crystal CCS thus correlates with the CCS of the 11+ SPM-1 ion and the ‘closed’ form matched the CCS of the 10+ charge state, suggesting the presence of both forms.


Studying the active-site loop movement of the São Paolo metallo-β-lactamase-1†Electronic supplementary information (ESI) available: Procedures for protein expression and purification, (19)F-labelling, crystallisation, data collection, and structure determination, table of crystallographic data, table of crystallographic parameters and refinement statistics, figures showing binding mode and distances, procedures for mass spectrometry measurements, differential scanning fluorimetry measurements, stopped-flow measurements and other kinetics measurements. See DOI: 10.1039/c4sc01752hClick here for additional data file.

Brem J, Struwe WB, Rydzik AM, Tarhonskaya H, Pfeffer I, Flashman E, van Berkel SS, Spencer J, Claridge TD, McDonough MA, Benesch JL, Schofield CJ - Chem Sci (2014)

Non-denaturing ion mobility MS reveals different conformations of di-Zn(ii)-SPM-1 as compared to the theoretical collisional cross section (CCS) values of the ‘open’ and ‘closed’ crystal forms.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4333608&req=5

fig3: Non-denaturing ion mobility MS reveals different conformations of di-Zn(ii)-SPM-1 as compared to the theoretical collisional cross section (CCS) values of the ‘open’ and ‘closed’ crystal forms.
Mentions: IM-MS was used to investigate different conformational states of SPM-1 with and without Zn bound through reporting their corresponding CCS values. An IM-MS spectrum under conditions that preserve non-covalent interaction shows three major charge states (9+ → 11+) of SPM-1 with distributions of z = 9+ (10%), 10+ (60%), 11+ (30%), corresponding to a mass of 28 030.5 Da (Fig. 2A). The CCS of the charge states are: 2119 Å2 (9+), 2167 Å2 (10+) and 2247 Å2 (11+) (Fig. 3B). The calculated CCS from X-ray structures of ‘open’ and ‘closed’ forms of SPM-1 are 2237 Å2 (open) and 2171 Å2 (closed). The ‘open’ crystal CCS thus correlates with the CCS of the 11+ SPM-1 ion and the ‘closed’ form matched the CCS of the 10+ charge state, suggesting the presence of both forms.

Bottom Line: These include a mobile α3 region which is important in catalysis and determining inhibitor selectivity.SPM-1 thus appears to be a hybrid B1/B2 MBL.The results have implications for MBL evolution and inhibitor design.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry , University of Oxford , 12 Mansfield Road , Oxford , OX1 3TA , UK . Email: christopher.schofield@chem.ox.ac.uk.

ABSTRACT

Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibiotics. We report biophysical and kinetic studies on the São Paulo MBL (SPM-1), which reveal its Zn(ii) ion usage and mechanism as characteristic of the clinically important di-Zn(ii) dependent B1 MBL subfamily. Biophysical analyses employing crystallography, dynamic (19)F NMR and ion mobility mass spectrometry, however, reveal that SPM-1 possesses loop and mobile element regions characteristic of the B2 MBLs. These include a mobile α3 region which is important in catalysis and determining inhibitor selectivity. SPM-1 thus appears to be a hybrid B1/B2 MBL. The results have implications for MBL evolution and inhibitor design.

No MeSH data available.