University of Groningen

Ntn-hydrolases unveiledBokhove, Marcel

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Citation for published version (APA):Bokhove, M. (2010). Ntn-hydrolases unveiled: structural investigations into isopenicillin N acyltransferaseand the quorum-quenching acylase PvdQ. Groningen: s.n.

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Chapter 2

Crystallization of the quorum-quenching N-acyl homoserinelactone amidohydrolase PvdQ from Pseudomonas aeruginosa

Bokhove M, Nadal Jimenez P, Quax WJ & Dijkstra BW

Chapter 2

Abstract

P vdQ is a quorum-quenching N-acyl homoserine lactone amidohydrolase, or

acylase, from Pseudomonas aeruginosa that is capable of disrupting bacterial

communication, also called quorum sensing. In order to determine the structural

details that underlie quorum quenching, PvdQ was crystallized at 293 K, using 21 - 24 %

PEG 6000 in 100 mM BICINE buffer, pH 9.1. Crystals were of space group C2221 with

unit cell parameters a = 120.1 Å, b = 163.9 Å, c = 93.6 Å, α = β = γ = 90.0◦ and

showed diffraction to 1.8 Å resolution on the ID14-2 beamline of the European Synchrotron

Radiation Facility in Grenoble, France. Here we present the initial steps towards the crystal

structure determination of an N-acyl homoserine lactone acylase.

28

Crystallization of Pseudomonas aeruginosa PvdQ

Introduction

In many Gram-negative pathogens virulent behavior is regulated by quorum sensing, in

which N-acyl homoserine lactones (AHLs) act as diffusible messaging compounds (for a

review see e.g. Whitehead et al., 2001). In recent years enzymes have been discovered

that are capable of disrupting quorum sensing by breaking down these AHLs, such that

they can no longer be used as signaling molecules. Quorum sensing signal disruption,

also called quorum quenching, can be realized by hydrolysis of either the ester bond in

the homoserine lactone ring (performed by lactonases) (Dong et al., 2001), or the peptide

bond that connects the acyl chain and the homoserine lactone core (performed by acylases)

(Huang et al., 2003; Sio et al., 2006). In the light of the increase in bacterial drug resistance

quorum quenching has gained interest as a potential tool in the development of novel

antimicrobial strategies (Hentzer & Givskov, 2003). Crystal structures of such enzymes

pinpoint the residues involved in substrate recognition; structure based mutations could

be used to modify substrate specificity towards different N-acyl substituted homoserine

lactones from different pathogenic organisms. A structurally well-characterized quorum-

quenching enzyme is the N-acyl homoserine lactone lactonase from Bacillus thuringiensis

(Kim et al., 2005; Liu et al., 2005), but as of yet no structural data is available for an N-

acyl homoserine lactone acylase. Here we report the initial steps towards the first crystal

structure elucidation of a quorum-quenching N-acyl homoserine lactone acylase, PvdQ

from Pseudomonas aeruginosa.

Materials and methods

1. Purification of PvdQ

PvdQ was cloned and overexpressed according to Sio et al. (2006). The cell-pellet was

resuspended in three volumes of lysis buffer (50 mM TRIS-HCl, pH 8.8, 2 mM EDTA),

lysed by sonication and centrifuged at 30,000 g to remove cell debris. The supernatant

was applied to a HiTrap Q-Sepharose column (GE Healthcare Life Sciences, Uppsala,

Sweden). PvdQ appeared in the flow-through. After bringing the flow-through fraction to

700 mM ammonium sulfate, the protein solution was applied to a HiTrap phenylsepharose

column (GE Healthcare Life Sciences). PvdQ eluted at the end of a 700-0 mM ammonium

sulfate gradient. Finally, PvdQ was concentrated to 4 mg/ml and applied to a Hiload

Superdex75 16/160 gel filtration column (GE Healthcare Life Sciences) and the major peak

29

Chapter 2

was collected. Purified PvdQ was analyzed by SDS PAGE and by dynamic light scattering

using a DynaPro MSTC161 apparatus (Wyatt Corporation, Santa Barbara, USA).

2. Thermal Shift Assay and Buffer Exchange

Since PvdQ was purified without any additives during hydrophobic interaction and ion-

exchange chromatography, the thermal shift assay (Ericsson et al., 2006) was used to

establish a protein buffer capable of stabilizing PvdQ during storage and crystallization. The

thermal shift experiment was performed in a 96 well format on a MyiQ rtPCR apparatus

(Bio-Rad, Hercules, USA), in which a linear temperature gradient from 293 to 373K was

used to unfold the protein. The fluorescence of Sypro orange (Bio-Rad) was used as a

marker for protein unfolding. Sypro orange was excited at 485 nm and the fluorescence

was detected at 575 nm. Screening for stabilizing conditions was done over a pH range

from 4 to 11 using the MMT buffer system (Newman, 2004) supplemented with different

additives including: 5 % glycerol, 100 mM NaCl, a combination of glycerol and NaCl, 2

mM DTT, 5 mM CaCl2 5mM MgCl2, 25 mM NaAc or a combination of NaCl, CaCl2 and

DTT. Protein melting curves were analyzed using the software supplied by the vendor. The

melting temperature (Tm) of PvdQ in the presence of different additives was plotted against

the pH. The Tm is considered to be related to the protein stability (Ericsson et al., 2006).

3. Crystallization

The purified protein was concentrated to 7 mg/ml and transferred to the stabilization

buffer obtained from the thermal shift assay, using an Amicon centrifugal filtration device

with a 50 kDa molecular mass cut-off. To obtain suitable crystallization conditions for

PvdQ, several screens were set up in sitting drop plates with an Oryx-6 crystallization

robot (Douglas Instruments, Hungerford, UK). These screens included Structure Screen

(Molecular Dimensions, Newmarket, UK), Wizard screen (Emerald Biosystems, Bainbridge

Island, USA), JCSG+ (Qiagen, Valencia, USA) and PACT premier (Molecular Dimensions).

Drops were prepared by mixing 180 nl protein solution with 120 nl well solution. The

drops were subsequently equilibrated against 75 µl well solution at 293 K. Crystals

appeared overnight in a solution containing 100 mM CHES buffer, pH 10.0, 20 % PEG

8000 (Wizard screen). These crystals were further optimized in a hanging drop setup

using custom-made plates by mixing 1 µl protein solution and 1 µl reservoir solution, and

allowing vapour diffusion against 500 µl well solution.

30

Crystallization of Pseudomonas aeruginosa PvdQ

4. Data Collection and analysis

Cryo-crystallographic diffraction data were collected at the European Synchrotron Radiation

Facility (ESRF) in Grenoble, France. PvdQ crystals were mounted in Hampton Research

(Aliso Viejo, USA) cryoloops and cryoprotected in mother liquor supplemented with 25 %

glycerol and flash-frozen in liquid nitrogen. Data were collected at the ESRF beamline

ID14-2 at a wavelength of 0.933 Å on a Quantum 4R CCD area detector (ADSC, Poway,

USA). Data were integrated and scaled with XDS (Kabsch, 1993) and merged with SCALA

(Evans, 2006).

Results and discussion

Purified PvdQ shows bands of 19 and 60 kDa on SDS-PAGE gels corresponding to the α-

and β-subunits, respectively, (Figure 1A). Dynamic Light Scattering experiments indicated

that PvdQ is monodisperse in solution with a particle size corresponding to a molecular

weight of approximately 80 kDa (results not shown).

A B

β

α

976645

30

20

14

Thermal shift assay on PvdQ

4647484950515253545556

4.0 5.0 6.0 7.0 8.0 9.0pH

T m

Glycerol

NaCl

Glycerol/NaCl

DTT

CaCl2

MgCl2

DTT/Glycerol/CaCl2

NaAc

Figure 1: (A) SDS PAGE gel of PvdQ, which clearly shows that PvdQ is a heterodimer with an α- and a β-subunit.The right lane contains the molecular weight markers. (B) Thermal shift assay showing that PvdQ is most stable inTRIS-HCl buffer, pH 7.5, and 5 % glycerol.

To find a suitable buffer for storage and crystallization, different pHs and additives were

screened using the thermal shift assay. Figure 1B shows a plot of the Tm plotted against the

pH, with every line corresponding to a different additive. From this plot it is very clear that

5 % glycerol has the strongest stabilizing effect. Consequently, a buffer containing 50 mM

TRIS-HCl, pH 7.5, and 5 % glycerol was used for storage and crystallization of PvdQ.

31

Chapter 2

PvdQ could be crystallized in a condition of the Wizard Screen (Emerald Biosystems)

consisting of 100 mM CHES buffer, pH 10.0, 21 - 24 % PEG 8000. Optimized

crystallization conditions were obtained using 100 mM BICINE buffer, pH 9.1, and 23 %

PEG 6000. Crystals grew to dimensions of approximately 50 x 50 x 200 µm (Figure 2A).

Data to 1.8 Å were collected on the ID14-2 beamline at the ESRF. A typical diffraction

pattern is shown in Figure 2B.

Crystals belonged to space group C2221 with unit cell parameters a = 120.1 Å,

b = 163.9 Å, c = 93.6 Å, α = β = γ = 90.0◦. Data collection statistics can be found in

Table 1. Analysis of the data indicated that the crystal contained one 80 kDa heterodimeric

molecule per asymmetric unit with a Matthews coefficient of 2.9 Å3 Da-1 and a solvent

content of 57 % (Matthews, 1968).

A B

1.8 Å

Figure 2: (A). Several single crystals of PvdQ obtained in 100 mM BICINE buffer, pH 9.1, and 20 - 24 % PEG 6000.(B) A typical diffraction pattern from a PvdQ crystal. The edges of the detector correspond to 1.8 Å resolution.The inset shows an enlarged view of the bottom right corner of the detector (Å).

Initial molecular replacement runs were performed with PHASER (McCoy et al., 2005)

using an ensemble consisting of cephalosporin acylase (PDB entry 1KEH) (Kim et al., 2002)

and penicillin G acylase (PDB entry 1E3A) (Hewitt et al., 2000), which were found using

the Fold & Function Assignment System (FFAS) (Jaroszewski et al., 2005). The conserved

residues were kept, while variable residues were replaced with serine residues using the

SCWRL modeler (Canutescu et al., 2003). Initial molecular replacement rounds resulted

in a rotation function Z-score of 6.6, a translation function Z-score of 8.9 and a log-

likelihood gain of 96. Chapter 3 discusses the structure elucidation of several PvdQ ligand

complexes and a Serβ1Cys mutant impaired in catalysis.

32

Crystallization of Pseudomonas aeruginosa PvdQ

Table 3: Data collection of wild type, apo-PvdQ. Values in parentheses correspond to the high-resolution shell.

Parameter ValueData collection

Beam line ESRF ID14-2Wavelength (Å) 0.933Temperature (K) 100Detector Quantum 4R CCD (ADSC)

Data ProcessingSpace group C2221

Unit cell parameters (Å, ◦) a = 120.1, b = 163.9, c = 93.6;α = β = γ = 90

Resolution (Å) 40.0-1.8 (1.9-1.8)Total reflections 348,157 (49,902)Unique reflections 85,309 (12,328)Redundancy 4.1 (4.0)I/σ(I) 24.4 (4.4)Rsym

¶ (%) 3.8 (34.0)Completeness (%) 99.7 (99.7)Vm (Å3 kDa-1) 2.9Solvent content (%) 57

¶Rsym =

∑hkl

∑i |Ii(hkl) − I(hkl)|∑hkl

∑i Ii(hkl)

33