SUPPORTING INFORMATION
CRYSTAL STRUCTURE OF NEW DELHI METALLO--LACTAMASE (NDM-1)
REVEALS MOLECULAR BASIS FOR ANTIBIOTIC RESISTANCE
Dustin King, Natalie Strynadka*
From The Department of Biochemistry and Molecular Biology and Center for Blood Research,
University of British Columbia, Vancouver, British Columbia
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SUPPORTING MATERIALS AND METHODS
This section outlines materials and methods used in supplementary figures 1 and 2.
ICP Mass Spectrometry
Inductively coupled mass spectrometry (ICP-MS) was used to analyze purified NDM-1
samples for the presence of various metal ions (Mg24, Al27, Mn55, Co59, Ni60 and Zn66). The
NDM-1 protein was purified as previously described and freshly dialyzed into ms-buffer (20mM
HEPES, 100mM NaCl, pH7) in order to remove any contaminating metals. The protein was
concentrated to ~12mg/mL and diluted 1/100 in ms buffer, followed by a 1/10 dilution in an
internal standard (10ug/L Sc45, 1% nitric acid, Inorganic Ventures). Prior to sample analysis, the
ICP MS was calibrated using a standard solution containing the metal isotopes of interest
(Inorganic Ventures). The protein sample was then transferred by nebulization into a NexION
3000 ICP mass spectrometer (Perkin Elmer). Quantitative analysis was performed in triplicate
for each sample with 60 sweeps per reading using the peak-hopping mode with a 50ms/AMU
dwell time for each element. Instrument settings were: rf power (1600 W), integration time (35s),
collision gas (Ar40), RPQ voltage (25V) and sample flow rate (4 rpm). Isotope abundance was
determined by integrating peak areas using the NexION software program, and the data was
represented graphically using Prism.
Dynamic Light Scattering
Full length NDM-1 protein was purified as previously outlined, concentrated to
~10mg/mL and double filtered through a 0.22-µM filter prior to analysis.
Samples were
immediately added to a clean quartz cuvette with a 1L inner volume and analyzed using a
DynaPro nanostar instrument (Wyatt). The light source for the system was a 100 mW air
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launched laser, operating at 662 nm. All measurements were made at a constant temperature of
21°C, path length of 1cm and a scattering angle of 90°.
Data were fit using the Wyatt
DYNAMICS software program to attain an approximate hydrodynamic radius and corresponding
molecular weight.
Chemical Cross Linking
Purified full-length NDM-1 was prepared as previously described. Purified protein was
exchanged into fresh cross-linking buffer (20mM HEPES, 150mM NaCl, pH8.0). A total of
140g of purified NDM-1 protein was added to varying amounts of the chemical cross linker bis
[sulfosuccinimidyl] suberate (Thermo Scientific). Reactions were made up to a final volume of
30L with cross-linking buffer and incubated at room temperature for 30 min. The reactions
were terminated using quenching buffer (1M Tris-HCl, pH 7.5) and immediately prepared for
SDS polyacrylamide gel electrophoresis, or western blot analysis.
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SUPPORTING TABLES
Supporting Table S1. Data collection and refinement statistics for NDM-1
Data collection
Wavelength
Resolutiona
NDM-1 apo
CLS CMCF-2
1.00 Å
49.21-2.10 Å (2.212.10)
P1
66.536 Å
73.904 Å
77.407 Å
70.32, 75.86, 65.30°
.083(.533)
9(1.8)
96%(89.5%)
70204(9574)
2.2(2.2)
Space group
a
b
c
α, β, γ
Rsym (%)a
I/σ(I)a
Completenessa
Unique reflectionsa
Redundancya
Refinement
Average B factor (Å2)
Protein
28.2
Ligand
30
Water
32
Ramachandran statistics
Favored regions
92.2%
Additionally allowed regions
7.3%
Disallowed regions
0.5%
Rwork
21.7%
Rfree
25.1%
r.m.s.b bonds
0.014Å
r.m.s. angles
1.86°
a
Values in parenthesis represent the highest resolution shell.
b
r.m.s. means root mean square.
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SUPPORTING FIGURES
Supporting Figure S1. The oligomeric state of NDM-1. A, The size exclusion chromatography
profile of FL NDM-1. The corresponding peaks are marked, along with appropriate positions of
molecular mass standards 67 and 47 kDa. B, Dynamic light scattering of FL NDM-1. The
monomer and dimer fractions, collected from gel filtration are shown in red and blue. The plot
indicates that NDM-1 can exist in both monomeric and dimeric forms in solution. The results are
displayed as particle size distributions with the inset showing the results summary table. C, 12%
SDS-PAGE gel analysis of chemical cross linking between NDM-1 dimers. Samples were
incubated with and without 2.5 mM bis [sulfosuccinimidyl] suberate (BS3) chemical cross linker.
The negative control (IMP-1) is a MBL that is known to be predominantly monomeric in
solution.41
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Supporting Figure S2. NDM-1 metal ion analysis by ICP mass spectrometry. Zinc was the
only metal ion present in high abundance, with a Zn/NDM-1 protein ratio of ~1.53.
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