Crystal structures of KPC-2 β-lactamase in complex with 3-nitrophenyl boronic acid and the penam sulfone PSR-3-226

Article abstract of DOI:10.1128/AAC.06099-11

Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-A resolution. 3-NPBA demonstrated a K_m value of 1.0±0.1 μM (mean±standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-A resolution. PSR-3-226 displayed a K_m value of 3.8±0.4 μM for KPC-2, and the inactivation rate constant (k _inact) was 0.034±0.003 s^-1. When covalently bound to S70, PSR-3-226 forms a trans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first β-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors. Copyright

Full text of DOI:10.1128/AAC.06099-11

Crystal Structures of KPC-2 ␤-Lactamase in Complex with
3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226
Wei Ke,a Christopher R. Bethel,f Krisztina M. Papp-Wallace,e,f Sundar Ram Reddy Pagadala,g,h Micheal Nottingham,g,h
Daniel Fernandez,g,h John D. Buynak,g,h Robert A. Bonomo,b,c,d,e,f and Focco van den Akkera
Departments of Biochemistry,a Pharmacology,b Molecular Biology and Microbiology,c and Medicinee and Center for Proteomics,d Case Western Reserve University,
Cleveland, Ohio, USA; Research Service, Louis Stokes Cleveland VA Medical Center, Cleveland, Ohio, USAf; Department of Chemistry, Southern Methodist University, Dallas,
Texas, USAg; and The SMU Center for Drug Discovery, Design, and Delivery at Dedman College, Dallas, Texas, USAh
Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is
not easily inhibited by ␤-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mecha-
nisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two ␤-lactamase inhibitors that follow differ-
ent inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid
(3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated a Km value of 1.0 ؎ 0.1 ␮M (mean ؎ standard error)
for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron
hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the
aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the pe-
nam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed a Km value of 3.8 ؎ 0.4 ␮M for KPC-2, and the
inactivation rate constant (kinact) was 0.034 ؎ 0.003 s؊1. When covalently bound to S70, PSR-3-226 forms a trans-enamine inter-
mediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides
in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226
are the first ␤-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights
gained here could aid in the design of potent KPC-2 inhibitors.
arbapenemases pose a serious clinical threat to the “last-resort 226 is a derivative of SA2-13 (Fig. 1) with a different C-2 moiety.
C
antibiotics,” the carbapenems (imipenem, meropenem, er- Previously, SA2-13 was rationally designed to stabilize the inhibitory
tapenem, and doripenem) (22, 33). In particular, the Klebsiella trans-enamine intermediate (16). We present here the first structures
pneumoniae carbapenemases (KPCs) are troublesome, as they are of ␤-lactamase inhibitors in complex with KPC-2. The knowledge
often carried on a plasmid, leading to rapid dissemination (14, 22, obtained from this structural analysis is a starting point for structure-
30, 33). The first isolated member of the KPC family, KPC-2 ␤-lac- based inhibitor optimization.
tamase, was identified in 1996 in North Carolina as part of the
Intensive Care Antimicrobial Resistance Epidemiology (ICARE)
Project (36). To date, 11 KPC variants have been described (http:
//www.lahey.org/studies/), with KPC-2 and KPC-3 being the
dominant variants; both KPC-2 and KPC-3 are becoming en-
demic in the United States, Greece, and Israel (35). KPCs have also
MATERIALS AND METHODS
Cloning. A minor truncation of the C terminus of KPC-2 resulted in im-
proved crystal growth (25). We therefore engineered a similar truncated
KPC-2 construct by removing the last 4 KPC-2 amino acid residues by using
the following primers: forward primer, 5=-GAATTCCATATGTCACTGTA
emerged in China, South America, and many countries in Europe TCGCCGTCTAGTT-3=; reverse primer, 5=-CCGGAATTCTTAGCCCA
ATCCCTCGAG-3=. The gene encoding the truncated KPC-2 was PCR
(35). Although Klebsiella pneumoniae is the predominant host
amplified using Pfx polymerase (Invitrogen) and the pBR322-catI-
species for KPC ␤-lactamases, other Enterobacteriaceae, Pseu-
blaKPC-2 vector template, gel purified using a QIAquick gel extraction kit
(Qiagen), and ligated into the NdeI and EcoRI restriction sites of the
pET30a vector (Novagen) prior to transformation into Escherichia coli
DH5␣ cells (36). After sequence confirmation, the plasmid was trans-
formed into E. coli BL21(DE3) competent cells (Invitrogen) for large-scale
protein expression.
domonas spp., and Acinetobacter spp. have recently been reported
to harbor blaKPC genes (6, 23, 27, 32, 35). These reports represent
a disturbing development in the spread of these carbapenemases.
Microbiological and biochemical studies have shown that KPCs
are only weakly inhibited by clavulanic acid, sulbactam, and tazobac-
tam (14, 18), spurring the need for development of inhibitors against
this ␤-lactamase (7, 8, 25). A number of approaches exploiting dif-
ferent inhibitor scaffolds are being undertaken to find inhibitors of
class A ␤-lactamases (3). In this report, we focus on two of these
scaffolds: boronic acid compounds, which are used as probes to un-
derstand structural interactions in class A and C ␤-lactamases and in
KPC detection assays (2, 4, 9, 21), and 6-(unsubstituted)penam sul-
fones, which have been shown to inhibit KPC-2 in the low micromo-
lar range (18). We determined the structure of KPC-2 in complex
with 3-nitrophenyl boronic acid (3-NPBA) and with PSR-3-226 (Fig.
1) and tested each compound’s activity against KPC-2 (26). PSR-3-
Expression and purification. Six liters of lysogeny broth (LB) con-
taining 30 ␮g/ml kanamycin was inoculated with 3% overnight culture,
Received 8 November 2011 Returned for modification 25 December 2011
Accepted 4 February 2012
Published ahead of print 13 February 2012
Address correspondence to Focco van den Akker, focco.vandenakker@case.edu.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.06099-11
0066-4804/12/$12.00 Antimicrobial Agents and Chemotherapy p. 2713–2718
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Ke et al.
FIG 1 Chemical structures of the inhibitors 3-NPBA, PSR-3-226, and SA2-13.
and cultures were grown at 37°C until the optical density at 600 nm 6,000 Da) or polyethylene glycol 6000 (PEG 6000) in 100 mM potassium
reached 0.5 to 0.6. Subsequently, the temperature was lowered to 20°C, thiocyanate (KSCN) and 100 mM citrate (pH 4) at 20°C by using the
expression was induced with 0.4 mM isopropyl-␤-D-thiogalactopyrano- vapor diffusion sitting drop method with a drop size of 1 ␮l protein and a
side (IPTG; Sigma), and growth was allowed to continue overnight. Cells 1 ␮l reservoir. To obtain an inhibitor-complexed KPC-2 structure, we did
were subsequently pelleted by centrifugation and stored at Ϫ80°C until not include citrate buffer in the soaking and freezing solutions, since ci-
further use. Cell pellets were thawed and resuspended in 10 mM Tris trate might compete with the inhibitor in the active site (25). The crystals
buffer (pH 7.0) followed by lysis using sonication. After centrifugation, were therefore soaked overnight in a 3-NPBA soaking solution (contain-
the filtered supernatant was passed through a preequilibrated phenylbo- ing 50 mM 3-NPBA in 25% PEG 6000 and 100 mM Tris-Cl [pH 7.2]) and
ronate affinity column as previously described (8). Briefly, a phenylboro- were subsequently used for data collection. A similar soaking approach
nate affinity column was preequilibrated with 10 mM Tris buffer (pH 7.0). was attempted to obtain a PSR-3-226:KPC-2 structure, but this approach
The column was washed with 10 mM Tris buffer (pH 7.0)–0.5 M NaCl was not successful, as the crystals dissolved. To obtain the PSR-3-226-
before eluting KPC-2 with 0.5 M boronate (pH 7.0)–0.5 M NaCl. KPC-2 bound structure, KPC-2 crystals were soaked in a solution containing 50
was further purified using a Superdex 75 size exclusion column (GE Bio- mM PSR-3-226 with 25% PEG 6000 and 100 mM citrate (pH 4.0) for 2 h
sciences) equilibrated in 40 mM bis-Tris (pH 5.9). Sodium dodecyl sul- 45 min (longer soaking periods caused the crystal to visibly deteriorate).
fate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated The soaked crystals were cryo-protected with 20% ethylene glycol in the
that the homogeneity of KPC-2 was over 95%. The protein was subse- corresponding mother liquor inhibitor soaking solution and flash-frozen
quently concentrated to 22 mg/ml in 40 mM bis-Tris (pH 5.9). About 60 in liquid nitrogen prior to data collection.
mg of purified KPC-2 protein was obtained from a 6-liter culture.
Data collection and structure determinations. Data for the KPC-2:
Synthesis of PSR-3-226. PSR-3-226 was prepared as part of a large li- 3-NPBA and the KPC-2:PSR-3-226 complexes were collected at the Stan-
brary of more than 50 2=-substituted 6-(unsubstituted)penam sulfones, ford Synchrotron Radiation Lightsourse (SSRL) beamline BL11-1 and
which were synthesized and screened for activity against 8 different ␤-lacta- Advanced Photon Source (APS) beamline 23-ID, respectively. Both data
mases (17). These data, including the synthesis and structure-activity rela- sets were processed using HKL2000 (15). The KPC-2 complex structures
tionships (SARs), will be published separately. The synthetic scheme illustrat- were determined using molecular replacement with the program Phaser
ing the methodology utilized in this library design is illustrated in Fig. 2.
(11) with chain A of the truncated KPC-2 structure (PDB 3C5A) (25) as
Crystallization and soaking experiments. Initial crystallization con- the search model. Crystallographic refinement was performed using
ditions were obtained using sparse matrix crystallization screens and REFMAC (12), and model building was done using COOT (5). The
tested under the previously published KPC-2 crystallization conditions PRODRG2 server (29) was used to obtain the parameter and topology files
(8, 25). The best crystals were grown from 20% polyethylene glycol (Mw, for the chemical structures built in the active site, including the 3-NPBA
FIG 2 Synthesis of PSR-3-226. Reagents and conditions were as follows: (a) H2SO4, NaNO2, KBr, ethanol (EtOH), 6 to 8°C, 3.5 h; (b) peracetic acid,
benzophenone hydrazone, KI, DCM/H2O, 0°C, 1 h; (c) Zn dust, AcOH/CH3CN, 0°C, 1 h; (d) MBT, toluene, 114°C, 2 h; (e) ClCH2CO2H, AgOAc, DCM, room
temperature (RT), 4 h; (f) KMnO4, AcOH-H2O-acetone (5:1:1), RT, 1 h; (g) thiourea, EtOH, 70°C, 1 h; (h) DMP, DCM, RT, 1 h; (i) NaClO2, NaH2PO4,
3-methyl-2-butene, tBuOH-THF (3:1), RT, 1 h; (j) oxalyl chloride, pyr, DCM, RT, 1 h; (k) HOCH2CONH2, pyr, DMAP; (l) (i) H2, cat. Pd and (ii) NaHCO3 or
(i) TFA, anisole, Ϫ10°C and (ii) NaHCO3.
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Crystal Structures of KPC-2 ␤-Lactamase and Inhibitors
TABLE 1 Data collection and refinement statisticsa
TABLE 2 Kinetics of inhibitiona
Study phase and
parameter
Complex
K
m (␮M)
k
inact (sϪ1
ND
)
3-NPBA:KPC-2
P212121
PSR-3-226:KPC-2
P212121
KPC-2:3-NPBA
SHV-1:PSR-3-226
KPC-2:PSR-3-226
1.0 Ϯ 0.1
4.43 Ϯ 0.22
3.8 Ϯ 0.4
Data collection
Space group
Cell dimensions
a, b, c (Å); ␣, ␤,
␥ (°)
0.10 Ϯ 0.01
0.034 Ϯ 0.003
a Values are means Ϯ standard errors. ND, not determined.
50.4, 66.6, 73.6; 90, 90, 49.1, 66.3, 71.8; 90, 90,
90
90
Wavelength (Å)
Resolution (Å)
Rsym
0.97945
0.98400
hibitor concentrations. The kobs values were determined using a nonlinear
least squares fit of the data employing Origin 7.5 software and the follow-
ing equation: A ϭ A0 ϩ vf t ϩ (v0 Ϫ vf)[1 Ϫ exp(Ϫkobst)]/kobs, where A is
the absorbance, v0 (expressed as the variation of absorbance per unit of
time) is the initial velocity, vf is the final velocity, and t is time.
50.00–1.62 (1.68–1.62) 50.00–1.26 (1.31–1.26)
5.9 (36.1)
21.3 (3.3)
91.9 (75.7)
4.4 (4.4)
7.3 (27.2)
23.1 (3.4)
97.9 (82.5)
6.7 (2.7)
I/␴I
Completeness (%)
Redundancy
Next, kobs was plotted versus the inhibitor concentrations of the ex-
periment and fit to determine kinact according to the following equation:
Refinement
Resolution range (Å) 27.8–1.62 (1.661–1.619) 33.2–1.26 (1.293–1.260)
kobs ϭ (kinact[I]/(KI ϩ [I]).
No. of reflections
R(work)/R(free)
No. of atoms
Protein, inhibitor, 2,010, 12, 246, 0
water, others
RMSDb
Bond length (Å)
28,074
16.0/19.2 (25.8/29.2)
59,322
15.4/16.9 (22.5/24.6)
MICdeterminationsanddiskdiffusionassays. Thesusceptibilitiesof
Klebsiella pneumoniae possessing blaKPC-2 and E. coli DH10B pBR322-catI
expressing blaKPC-2 were assessed using Mueller-Hinton (MH) agar dilu-
tion MICs according to a method described by the Clinical and Labora-
tory Standards Institute (CLSI) (1). MICs were determined using a Steers
replicator, which delivered 10 ␮l of culture containing 104 CFU per spot.
The cefotaxime concentration was varied from 0.06 mg/liter to 128 mg/
liter, while 3-NPBA was maintained at 4 mg/liter. 3-NPBA and cefotaxime
were purchased from Sigma (St. Louis, MO).
2,013, 21, 316, 26
0.010
1.226
0.010
1.369
Bond angles (°)
Avg B factors (Å2)
Protein, inhibitor, 15.1, 22.5, 30.6, 0
water, others
Ramanchandran plot
statistics (%)
13.4, 53.0, 28.3, 73.8
Disk diffusion assays were performed following CLSI guidelines (13).
Disks containing 30 ␮g of cefotaxime were used alone (Becton Dickin-
son). Ten micrograms of 3-NPBA, 10 ␮g of PSR-3-226, and 30 ␮g of
PSR-3-226 were resuspended in dimethyl sulfoxide (DMSO) and pipetted
onto disks containing 30 ␮g of cefotaxime or blank (no inhibitor) disks
and allowed to dry for 1 h; in addition, DMSO alone was used as a control.
Colonies of E. coli DH10B that expressed pBR322-catI-blaKPC-2 were di-
rectly resuspended into MH broth in an amount producing the equivalent
of a 0.5 McFarland standard and were used to inoculate MH agar plates.
The disks were carefully placed on each plate. The bacteria were grown at
37°C for 18 h, and zone diameters were measured.
Core regions
94.7
94.7
5.3
Additional allowed 5.3
regions
Generously
0.0
0.0
0.0
allowed regions
Disallowed regions 0.0
a Values in parentheses refer to the highest-resolution shell.
b RMSD, root mean square deviation.
Protein Data Bank accession numbers. The coordinates and struc-
ture factors for the KPC-2 complexes have been deposited with the Pro-
tein Data Bank (PDBID for 3-NPBA:KPC-2, 3RXX; PDBID for PSR-3-
226:KPC-2, 3RXW).
and the trans-enamine intermediate of PSR-3-226. Crystallographic re-
finement was monitored using the program DDQ (31), and the final
model quality was assessed using PROCHECK (10). Data collection and
refinement statistics are shown in Table 1.
RESULTS AND DISCUSSION
MICs, kinetics, and the KPC-2:3-NPBA complex. In susceptibil-
ity testing, 3-NPBA at 4 mg/liter decreased cefotaxime MICs from
32 mg/liter to 4 mg/liter for K. pneumoniae and E. coli DH10B
strains expressing blaKPC-2. For E. coli DH10B carrying pBR322-
catI-blaKPC-2, zone diameters during disk diffusion assays in-
creased from 20 mm for cefotaxime alone to 37 mm for cefo-
taxime combined with 3-NPBA.
␤-Lactamase inhibition assays. Steady-state kinetic parameters were
determined with an Agilent 8453 diode array spectrophotometer at 25°C
in 10 mM PBS buffer using purified KPC-2. The kinetic parameters Vmax
and Km, calculated from initial steady-state velocities (v), were obtained
by using an iterative nonlinear least squares fit of the data to the Henri-
Michaelis equation and utilizing Enzfitter (Biosoft Corporation), accord-
ing to the following equation: v ϭ (Vmax[S])/(Km ϩ [S]), where v is the
observed velocity, Vmax is the maximum velocity, [S] is the substrate con-
centration, and Km is the Michaelis constant determined for nitrocefin.
For the purposes of these analyses, 3-NPBA and PSR-3226 were re-
garded as mechanism-based inhibitors. As a result, Km values of the in-
hibitors were determined in competition assays using 100 ␮M nitrocefin
(⌬␧482, 17,400 MϪ1 cmϪ1). Plots of 1/[S] versus activity values were linear
and provided the y intercept/slope values used for Km determinations. Km
values were corrected by taking into account the nitrocefin affinity, ac-
cording to the following equation: Km(corrected) ϭ Km(observed)/{1 ϩ
[S]/Km(nitrocefin)}, where [S] is the concentration of nitrocefin (100
␮M) and Km(nitrocefin) is the Michaelis constant determined for nitro-
cefin (for SHV-1, 20 ␮M; for KPC-2, 5 ␮M). The kinetic parameters were
calculated using data from experiments. The kinact values were determined
3-NPBA inhibited KPC-2 ␤-lactamase with a Km value of 1.0 Ϯ
0.1 ␮M (mean Ϯ standard error) (Table 2).
The X-ray crystal structure of KPC-2:3-NPBA was determined
to 1.62-Å resolution. During the initial model-fitting stages, an
unbiased omit Fo-Fc map contoured at 2.5␴ clearly revealed the
3-NPBA ligand covalently attached to the O␥ atom of the catalytic
S70 residues (Fig. 3A). Density for the nitro moiety of 3-NPBA is
somewhat weaker than that for the rest of 3-NPBA, which is per-
haps explained by the distance of this moiety from the boron-
carbon bond of 3-NPBA. A relatively small rotational heterogene-
ity around this bond would displace the more distant nitro group,
yielding a weaker density for this moiety than for the rest of
using a fixed concentration of enzyme and nitrocefin and increasing in- 3-NPBA. In addition to 3-NPBA, 246 water molecules were in-
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Ke et al.
FIG 3 Unbiased omit Fo-Fc electron density maps contoured at 2.5␴, depicting 3-NPBA in the active site of KPC-2 ␤-lactamase (A) or the trans-enamine
intermediate of PSR-3-226 in the active site of KPC-2 ␤-lactamase (B). Citrate molecules in panel B were omitted for clarity. (C) Partially occupied citrate
molecules in the PSR-3-226-bound KPC-2 structure.
cluded in the model, and the final model refined to an R(work) tion was described previously for larger boronic acid transition
value of 16.0% and an R(free) value of 19.2% (additional data and state inhibitor (BATSI) compounds when bound to SHV-1 ␤-lac-
refinement statistics are listed in Table 1).
tamase (9) (Fig. 4B). In this conformation, one of the boron hy-
The overall structure of KPC undergoes little change upon li- droxyl groups is positioned in the oxyanion hole and the other one
gand binding, as the 3-NPBA-bound KPC-2 structure was similar displaces the deacylation water positioned normally between E166
to that of the full-length KPC-2 and the C-terminally truncated and N170. A previous structure analysis of 3-NPBA bound to the
structures, yielding a root mean square deviation (RMSD) for C␣ class C ␤-lactamase AmpC (26) revealed a different orientation
atoms of 0.574 and 0.226 Å, respectively.
for the ligand compared to the KPC-2:3-NPBA structure. The
In Fig. 4, the interactions of 3-NPBA with the KPC-2 active site 3-nitro moiety of 3-NPBA in the AmpC complex is in a position
are illustrated. First, 3-NPBA forms a dative bond, via its boron that would not be compatible within the KPC-2 structure, as it
atom, with the O␥ atom of S70. Second, both boronate oxygens would sterically clash with the KPC-2 N170 side chain. One com-
form extensive interactions; one boronate oxygen hydrogen mon feature, however, between the two 3-NPBA structures is the
bonds with the backbone nitrogens of S70 and T237 and the car- phenyl ring-Asn interaction, as Asn152 of AmpC is in a similar
bonyl oxygen of T237, whereas the other boronate oxygen inter- position as Asn132 in KPC-2.
acts with the N170 and E166 side chains. Third, the CP5 carbon
atom of the phenyl ring of 3-NPBA undergoes a 3.8-Å van der diffusion assays, 10 ␮g of PSR-3-226 combined with cefotaxime
Waals interaction with W105 via an edge-to-face interaction, a did not change zone sizes of E. coli DH10B pBR322-catI-blaKPC-2
MICs, kinetics, and the KPC-2:PSR-3-226 complex. In disk
.
common interaction for aromatic moieties (1). Furthermore, the When 30 ␮g of PSR-3-226 was combined with cefotaxime, an
␲ ring electrons of the phenyl ring of 3-NPBA interact via cat- increase in zone size from 16 mm to 19 mm was observed.
ion-␲ interactions with K73 (4.0 Å) and N132 (3.6 Å). Both types
The measured affinities of PSR-3-226 for KPC-2 and SHV-1
of interactions with phenyl rings have been observed previously were similar, although it had a slightly lower Km for KPC-2 (Table
(24, 37). Finally, 3-NPBA allows an interaction with a water mol- 2). The inactivation rate constant kinact is, however, about 3-fold
ecule (Fig. 4A). The positions of the boronic acid oxygens of lower for KPC-2 compared to SHV-1, likely pointing to KPC-2’s
3-NPBA bound in the active site indicate that the inhibitor adopts inherent reduced susceptibility to inhibition.
a conformation that is reminiscent of a deacylation transition state
The X-ray crystal structure of the KPC-2:PSR-3-226 complex
inhibitor. Such a boronic acid transition state analog conforma- was determined to 1.26-Å resolution. The unbiased omit Fo-Fc
FIG 4 3-NPBA bound to KPC-2. (A) Stereo view of interactions of 3-NPBA in the active site of KPC-2 ␤-lactamase. (B) Active site superposition of the
KPC2:3-NPBA structure with the SHV-1:cefoperazone BATSI structure (PDB ID 3MKF). SHV-1:cefoperazone BATSI is shown in white, and the KPC-2:3-NPBA
structure is in black. Residues used for superpositioning include 70, 73, 105, 130, 132, 166, 170, and 234 to 237.
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Antimicrobial Agents and Chemotherapy

Crystal Structures of KPC-2 ␤-Lactamase and Inhibitors
FIG 5 PSR-3-226 bound to KPC-2. (A) Stereo view of interactions of PSR-3-226 in the active site of KPC-2. Citrate molecules were omitted for simplicity.
Dashed black lines indicated hydrogen bonds. (B) Active site superposition of the KPC-2:PSR-3-226 structure with the SHV-1:SA2-13 structure (PDB ID 2H5S).
KPC-2:PSR-3-226 is shown in black, while SHV-1:SA2-13 is white, with the protein shown in a transparent cartoon representation. Residues used for superpo-
sition are the same as those used in Fig. 4.
map contoured at 2.5␴ revealed density for a ligand covalently interactions were also observed for the SHV-1:SA2-13 complex.
attached to the O␥ atom of the catalytic S70 residue (Fig. 3B). A Furthermore, a superpositioning of these two structures revealed
trans-enamine intermediate was modeled into this density, and that substantial parts of SA2-13 and PSR-3-226 are in a similar
the dihedral angle for this trans-enamine was refined to 179.9°. conformation (Fig. 5B) (16). The interactions of the mostly dis-
Clear density for the atoms of this intermediate was observed up to ordered tail region of PSR-3-226 are not further discussed here,
its CB atom, whereas the electron density for the rest of the tail of due to the large uncertainty of their atomic positions and because
the compound was poor. This suggests that the tail region is flex- of their poor electron densities. In addition to PSR-3-226, there
ible, in particular around the CA-CB bond, and these poorly re- was also a partially ordered citrate molecule observed in the active
solved atoms include the sulfone moiety, methyl group, and site. The position of the carboxylate of citrate interacting with the
amide tail R2 linker. The apparent flexibility of this part of the KPC-2 active site was localized in the general carboxylate binding
compound was also evident from the refined higher B factors of site of serine ␤-lactamases, similar to where one of the other car-
these tail atoms, which is consistent with their poorly resolved boxylates of citrate, bicine, and even BLIP residue D49 were
density. Occupancy for the PSR-3-226 ligand was varied and shown to interact in earlier-described KPC-2 structures (7, 8, 25).
showed that the best refinement results occurred with an occu- The presence of a carboxyl moiety, (i.e., that of citrate), situated in
pancy of 0.7. Adjacent to PSR-3-226, a citrate molecule with two the carboxyl binding pocket in close proximity to the covalent
partial occupancies was also modeled, of which 5 citrate atoms O␥-carbonyl carbon bond, is reminiscent of SA2-13 bound to
had strong density (Fig. 3C). After careful examination and ad- SHV-1, in which SA2-13’s own carboxyl linker is situated in this
justment of the structure, we placed a water molecule with occu- position. Although we assigned this density as a partially ordered
pancy of 0.3 in the oxyanion hole and a deacylation water mole- citrate, we did consider the possibility that this density repre-
cule with 0.5 occupancy, bridging E166 and N170, to account for sented the amide linker of PSR-3-226 when in the trans-enamine
the extra density at these corresponding positions. In total, 316 intermediate conformation; this did not yield a satisfactory fit
water molecules were included in refinement, and the final model with the electron density, likely due to the linker being two atoms
had an R(work) of 15.4% and an R(free) of 16.9% (additional shorter than SA2-13. Therefore, this density was modeled as ci-
refinement statistics are listed in Table 1).
trate. Note that citrate was also present in the previous KPC-2
The overall protein conformation of KPC-2 when complexed crystal structure with one of its carboxyl moieties in a similar
to PSR-3-226 is similar to the uncomplexed full-length KPC-2 and position as in our structure (25). Interactions of the carboxyl moi-
the C-terminally truncated KPC-2 structures, with an RMSD of ety of citrate in the active site involved KPC-2 residues S130,
0.591 and 0.158 Å, respectively. The active site undergoes little adopting two different conformations, T235, T237, and K234.
change upon ligand binding, except for residues W105 and S130,
We did attempt to obtain a KPC-2:SA2-13 complex structure
which have two alternative conformations that appeared to corre- via soaking SA2-13 with KPC-2 crystals, but we were unsuccessful.
late with the 0.7 occupancy of PSR-3-226 and alternate conforma- This could have been due to steric repulsion, as superpositioning
tions of citrate, respectively. The alternate W105 conformations, of the SA2-13:wtSHV-1 structure with that of the PSR-3-226-
with 30 and 70% occupancies, are likely a result of avoiding steric bound KPC-2 structure indicated that SA2-13 binding to KPC-2
clashes with the inhibitor, which has 70% occupancy. PSR-3-226 would be sterically hindered due to W105 in either of the two
forms a linear trans-enamine intermediate in the active site, with observed conformations, as extrapolated van der Waals distances
the carbonyl oxygen being positioned in the oxyanion hole, as was would be less than 2.5 Å. We note that the region of the active site,
seen before for SA2-13 in SHV-1 (Fig. 5) and also in the R164 encompassing residues W105 and P104, has previously been ob-
mutants of SHV-1 (16, 28). Additional interactions are formed by served to have a key role in affecting substrate catalysis and inhi-
the C-3 carboxylate group of PSR-3-226, which is within hydro- bition efficacy in KPC-2 (19, 34).
gen bonding distance of N132, E166, and N170 (Fig. 5A). Such
In conclusion, 3-NPBA and PSR-3-226 are the first ␤-lacta-
May 2012 Volume 56 Number 5
aac.asm.org 2717

Ke et al.
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KPC-2. The KPC-2:PSR-3-226 structure presented here may serve
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a trans-enamine conformation suggests that this mode of inhibi-
tion has significant potential against KPC-2 ␤-lactamases, and the
structure provides insights into how to further stabilize this deacy-
lation-resistant intermediate.
The KPC-2:3-NPBA structure is also a potential starting point
for future drug design efforts to optimize boronic acid transition
state inhibitors. Such small boronic acid compounds have clear
potential as inhibitors, because similar compounds, such as
3-aminiphenylboronic acid, have a broad specificity for class A
carbapenemases (20). Further studies are under way to optimize
these types of inhibitors.
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KPC-2 beta-lactamase. Protein Sci. 19:1714–1727.
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boronic acid, dipicolinic acid and cloxacillin. Clin. Microbiol. Infect. 17:
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ACKNOWLEDGMENTS
J.D.B. is supported by the Robert A. Welch Foundation, grant N-0871.
F.V.D.A. is supported by the National Institutes of Health (R01
AI062968). The Veterans Affairs Merit Review Program, Geriatric Re-
search Education and Clinical Care (GRECC), and the National Institutes
of Health (RO1 AI063517-01) supported R.A.B. K.M.P.-W. is supported
by the Veterans Affairs Career Development Program.
The pBR322-catI-blaKPC-2 vector in Escherichia coli DH10B cells was a
kind gift of Fred Tenover, Centers for Disease Control and Prevention,
Atlanta, GA. We thank the staff of Stanford Synchrotron Radiation Light-
sourse beamline BL11-1 and Advanced Photon Source beamline 23-ID
for help with data collection.
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and prospects. Expert Rev. Anti Infect. Ther. 9:555–570.
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