The structure of the di-zinc subclass B2 metallo- bbbb...

1

The structure of the di-zinc subclass B2 metallo-ββββ-lactamase CphA reveals 1

that the second inhibitory zinc ion binds in the “histidine” site. 2

3

Carine Bebrone1,3,*,§

, Heinrich Delbrück2,*

, Michaël B. Kupper2, Philipp Schlömer

2, 4

Charlotte Willmann2, Jean-Marie Frère

1, Rainer Fischer

2, Moreno Galleni

3 and Kurt M. V. 5

Hoffmann2 6

7

1 Centre for Protein Engineering, University of Liège, Allée du 6 Août B6, Sart-Tilman 4000 8

Liège, Belgium 9

2 Institute of Molecular Biotechnology, RWTH-Aachen University, c/o Fraunhofer IME, 10

Forckenbeckstrasse 6, 52074 Aachen, Germany 11

3 Biological macromolecules, University of Liège, Allée du 6 Août B6, Sart-Tilman, 4000 Liège, 12

Belgium 13

14

* both authors contributed equally to this work 15

16

Running title: The inhibitory zinc ion binds in the “histidine” site of CphA 17

18

19

§ Corresponding author: Carine Bebrone, Centre for Protein Engineering, University of Liège, 20

Allée du 6 Août B6, Sart-Tilman, 4000 Liège, Belgium, Tel.: 003243663315, Fax: 21

003243663364, e-mail: [email protected] 22

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00288-09 AAC Accepts, published online ahead of print on 3 August 2009

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Abstract 23

Bacteria can defend themselves against β-lactam antibiotics through the expression of 24

class B β-lactamases, which cleave the β-lactam amide bond and render the molecule harmless. 25

There are three subclasses of class B β-lactamases (B1, B2 and B3), all of which require Zn2+ for 26

activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-β-lactamases 27

are most active as di-zinc enzymes, subclass B2 enzymes such as Aeromonas hydrophila CphA 28

are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to 29

determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals 30

allowed us to solve the crystal structures of the di-zinc forms of the wild-type enzyme and 31

N220G mutant. The first zinc ion binds in the “cysteine” site, as previously determined for the 32

mono-zinc form of the enzyme. The second zinc ion occupies a slightly modified “histidine” site, 33

where the conserved His118 and His196 residues act as metal ligands. This atypical coordination 34

sphere probably explains the rather high dissociation constant for the second zinc ion compared 35

to those observed in enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results 36

from immobilization of the catalytically-important His118 and His196 residues, as well as the 37

folding of the Gly232–Asn233 loop into a position that covers the active site. 38

39

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Introduction 41

Class B β-lactamases (also called zinc β-lactamases or metallo-β-lactamases) play a key 42

role in bacterial resistance to β-lactam antibiotics by efficiently catalyzing the hydrolysis of the 43

β-lactam amide bond. The existence of such enzymes is a particular concern because they are 44

effective against most β-lactam antibiotics (including the carbapenems), the corresponding genes 45

are easily transferred between bacteria and there are no clinically useful inhibitors. On the basis 46

of the known sequences, three different lineages, identified as subclasses B1, B2, and B3, can be 47

characterized (12, 13). All class B β-lactamases possess two potential zinc-binding sites and 48

share a small number of conserved motifs bearing some of the residues that coordinate the zinc 49

ion(s), notably His/Asn/Gln116-Xaa-His118-Xaa-Asp120 and Gly/Ala195-His196-Ser/Thr197 50

(13). Structural analysis of subclass B1 enzymes shows that one zinc ion has a tetrahedral 51

coordination sphere involving His116, His118, His196 and a water molecule or OH- ion 52

(“histidine” site), whereas the other has a trigonal-pyramidal coordination sphere involving 53

Asp120, Cys221, His263 and two water molecules (“cysteine” site) (Table 1). One 54

water/hydroxide serves as a ligand for both metal ions (3). In the mononuclear structures of B1 55

enzymes (BcII, VIM-2, SPM-1 and VIM-4), the sole metal ion was found to be located in the 56

“histidine” site (5, 15, 25 and P. Lassaux, unpublished data). In some monozinc B1 structures, the 57

Cys221 residue is found under an oxidized form. In subclass B3, the “histidine” site is similar to 58

that found in subclass B1, but Cys221 is replaced by a serine residue and the second zinc ion is 59

coordinated by Asp120, His121, His263 and the nucleophilic water molecule which again forms 60

a bridge between the two metal ions (Table 1) (33). 61

Aeromonas hydrophila CphA is a subclass B2 metallo-β-lactamase characterized by a 62

uniquely narrow specificity. CphA efficiently hydrolyses only carbapenems and shows very poor 63


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activity against penicillins and cephalosporins, in contrast to subclass B1 and B3 enzymes which 64

hydrolyse nearly all β-lactam compounds, with the exception of monobactams (10, 29). In 65

contrast to subclass B1 and B3 enzymes which are more active as di-zinc species, subclass B2 is 66

inhibited in a non-competitive manner by the binding of a second zinc ion. For CphA, the 67

dissociation constant of the second zinc ion (KD2) is 46 µM at pH 6.5 (16). In agreement with 68

EXAFS studies (17) and site-directed mutagenesis (34), the crystallographic structure of CphA 69

shows that the first zinc ion is in the “cysteine” site (14). However, the second binding site in 70

subclass B2 enzymes remains to be determined because Garau and colleagues could not produce 71

crystals of the di-zinc form despite presence of zinc concentration well above KD2 (14). The 72

structure of Sfh-1, a subclass B2 enzyme from Serratia fonticola, has been solved recently, once 73

again with only one zinc ion in the active site (11). In subclass B2, the His116 residue found 74

throughout the metallo-β-lactamase superfamily (with the exception of the B3 GOB enzymes 75

where Gln is present) is replaced by an asparagine (8, 12, 13). It has been previously shown that 76

the Asn116 residue has no role in the binding of the zinc ions in CphA (34). On the basis of 77

spectroscopic studies with the Aeromonas veronii cobalt-substituted ImiS enzyme, Crawford and 78

co-workers postulated that the second metal binding site was not the traditional “histidine” site 79

but a site, remote from the active site, which involves both His118 and Met146 as zinc ligands (6, 80

7). However, a recent study of potential zinc ligand mutants contradicts this hypothesis and 81

indicates that the position of the second zinc ion in CphA is probably equivalent to the “histidine” 82

site observed in subclasses B1 and B3 enzymes, with His118 and His 196 involved in the binding 83

of this second zinc perhaps with Cys221 (or Asn116 or Asp120) as the third ligand (4). 84

Since crystals of the wild-type CphA protein could not be obtained directly, single-site 85

mutants were engineered by site-directed mutagenesis and overproduced. These mutants were 86


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selected in order to introduce residues that are conserved in either subclass B1 or B3, or both (2). 87

Among these mutants, the N220G mutant (2) easily yielded crystals which were used as seeds to 88

grow wild-type CphA crystals (14). The kinetic parameters of the wild-type and N220G mutant 89

CphA enzymes were similar, indicating strong conservation of enzymatic properties in the mutant 90

(2). However, the N220G mutation results in a slightly higher KD2 value (86 µM) (2). We 91

therefore set out to solve the crystallographic structures of the di-zinc forms of the wild-type 92

CphA enzyme and its N220G mutant. We confirmed that the second zinc ion occupies a slightly 93

modified “histidine” site, where the conserved His118 and His196 residues act as metal ligands. 94

The implication of this discovery on what is known about the coordination of zinc ions by 95

metallo-β-lactamases is discussed. We propose an explanation for the inhibition by the second 96

zinc ion. Moreover, the role of the Asn233 residue in this inhibition phenomenon is also 97

apprehended based on a site-directed mutagenesis study. In this work, a structural role for the 98

zinc ions is also investigated. 99


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Materials and Methods 100

Protein purification and crystallization 101

Site-directed mutagenesis, protein expression and purification of the proteins were carried 102

out as described previously (2), with the addition of a third purification step consisting of a size 103

exclusion chromatography (Hiload 16/60 Superdex 75 prep grade column, Pharmacia Biotech). 104

The purified enzyme solution was dialyzed against 15 mM sodium cacodylate (pH 6.5). 105

Monodispersity of the protein solutions and the hydrodynamic radii of the dissolved protein 106

molecules were checked by Dynamic Light Scattering (DLS). The crystallisation conditions used 107

previously (14) were not easy to reproduce because the crystallisation drop comprised two 108

phases. Therefore, a screen was carried out to find new conditions. Initial screens were carried 109

out at 8°C using commercial Hampton Crystal Screens 1 and 2, Grid Screen Ammonium 110

Sulphate and Grid Screen PEG 6000 (Hampton Research, California, USA). N220G CphA (10 111

mg/ml) was crystallized from 2.2–2.4 M ammonium sulphate and 0.1 M MES (pH 6.0–6.5), 112

using the sitting drop method with new crystallisation plates designed by Taorad GmbH (Aachen, 113

Germany). The (1 µl) reservoir solution was mixed with the protein solution (1 µl) and the 114

mixture was left to equilibrate against the solution reservoir. Typically, orthorhombic crystals of 115

the N220G mutant grew within a few days to dimensions of 80 x 100 x 100 µm and belong to 116

space group C2221 (a=42.68, b=101.06 and c=116.82). Crystals of the wild-type CphA enzyme 117

were obtained under similar conditions using mutant micro-crystals as starting seeds and showed 118

similar orthorhombic symmetry in space group C2221 (a=42.80, b=101.50 and c=116.49). Before 119

data collection, 1µl of 100 mM ZnCl2 was added to the drops containing the wild-type and the 120

mutant crystals and soaked for one day. 121

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Data collection and processing 123

For data collection, wild-type and mutant crystals were transferred to a cryoprotectant 124

solution containing reservoir solution supplemented with 30% (v/v) glycerol. The mounted 125

crystals were flash-frozen in a liquid nitrogen stream. Near-complete X-ray data sets were 126

collected using a Bruker FR591 rotating anode X-ray generator and a Mar345dtb detector. 127

Diffraction data were processed with XDS (20) and scaled with SCALA from the CCP4-Suite 128

(32). 129

130

Structure determination and refinement 131

The structures of di-zinc variants from the wild-type and N220G mutant enzymes were 132

solved using a molecular replacement approach with Molrep (32), and the mono-zinc structure of 133

CphA (PDB file 1X8G) as starting model. The resulting model was rebuilt with ARP/wARP (28). 134

The structures were refined in a cyclic process including manual inspection of the electron 135

density with Coot (9) and refinement with Refmac (26). The refinement to convergence was 136

carried out with isotropic B-values and using TLS parameter (27). The positions of the zinc, 137

sulphate and chloride ions were verified by calculating anomalous maps and the occupancies of 138

the ions were determined by disappearing of difference electron density. Alternative 139

conformations were modelled for a number of side chains and occupancies were adjusted to yield 140

similar B-values for the disordered conformations. 141

142

Circular dichroism (CD) spectroscopy 143

Circular dichroism (CD) spectra for the apo-, mono-and di-zinc forms of the wild-type 144

and N220G enzymes (0.3 mg/ml) were obtained using a JASCO J-810 spectropolarimeter. The 145


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apoenzymes were obtained in the presence of 10 mM EDTA. The di-zinc forms were obtained in 146

the presence of 500 µM Zn2+. The spectra were scanned at 25°C with 1 nm steps from 200 to 250 147

nm (far UV). Thermal stability was assessed for the apo-, mono- and di-zinc forms of the wild-148

type and N220G proteins using CD spectroscopy at 220 nm with increasing temperature (25°C–149

85°C). Urea denaturation studies of the wild-type and N220G enzymes were also carried out in 150

the presence and absence of 500 µM Zn2+. The mono- and di-zinc enzymes were incubated for 151

18h in increasing concentrations of urea (0–8 M) at 4°C. Stability was assessed for the mono- and 152

di-zinc forms of both proteins using CD spectroscopy at 220 nm with increasing concentrations 153

of urea. 154

155

Thermal shift assay 156

Thermal shift assays were carried out using a LightCycler real-time PCR instruments 157

(Roche Diagnostics). Briefly, 10 µl of wild-type CphA enzyme or the N220G mutant (0.3 mg/ml) 158

was mixed with 10 µl of 5000 x Sypro Orange (Molecular Probes) diluted 1:500 in 15 mM 159

cacodylate (pH 6.5). Samples were heat-denatured from 25 to 90°C at a rate of 0.5°C per minute. 160

Protein thermal unfolding curves were monitored by detecting changes in Sypro Orange 161

fluorescence. The inflection point of the fluorescence vs temperature curves was identified by 162

plotting the first derivative over the temperature and the minima were referred to as the melting 163

temperature (Tm). Buffer fluorescence was used as the control. 164

165

Differential Scanning Calorimetry (DSC) 166

Thermal denaturation was investigated using a NDSC 6100 differential scanning 167

calorimeter (Calorimetry Sciences Corp., Lindon, USA) after dialysis of the N220G enzyme 168


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sample (1 mg/ml) against 15 mM cacodylate (pH 6.5). The dialysis buffer was used as reference. 169

The data were collected and analysed with a MCS Observer software package assuming a two-170

state folding mechanism. 171

172

Analysis of the N233A mutant 173

The Quik Change Site-directed mutagenesis kit (Stratagene Inc., La Jolla, Calif.) was used 174

to introduce the N233A mutation into CphA, using pET9a-CphA WT as the template, forward 175

primer 5′-GGAGAAGCTGGGCGCCCTGAGCTTTGCCG-3′ and reverse primer 5′-176

CGGCAAAGCTCAGGGCGCCCAGCTTCTCC-3′. The vector was then introduced into E. coli 177

strain BL21 (DE3) pLysS Star. Overexpression and purification of the mutant protein, CD 178

spectroscopy, electrospray ionization-mass spectrometry (ESI-MS) and the determination of 179

metal content, kinetic parameters and residual activity in the presence of increasing 180

concentrations of zinc were carried out as previously described (2, 4, 34). 181

182

Protein Data Bank accession codes 183

Coordinates and structure factors were deposited in the Protein Data Bank using accession 184

codes 3F90 (di- zinc form of wild-type CphA) and 3FAI (di-zinc form of the N220G mutant). 185


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Results 186

Structure determination. 187

The crystal structures of the zinc saturated wild-type and N220G CphA enzymes were 188

solved. Stereo-chemical parameters were calculated by PROCHECK (21) and WHAT_CHECK 189

(18) and fell within the range expected for a structure with similar resolution. The 190

crystallographic and model statistics for the di-zinc form of the wild-type and N220G mutant 191

structures are shown in Table 2. 192

193

Di-zinc form of the wild-type CphA metallo-ββββ-lactamase 194

The crystal structure of the di-zinc form of the wild-type CphA enzyme was solved by 195

molecular replacement using the structure of the mono-zinc form (PDB code 1X8G) as the 196

starting model. The structure was refined to a resolution of 2.03 Å. The Rwork and Rfree values for 197

the refined structure were 0.1589 and 0.2020, respectively. The crystals adopted a C2221 space 198

group with one molecule in the asymmetric unit. Only one residue (Ala195) was found in a 199

disallowed region of the Ramachandran plot. Ala195 (ψ= 105°, φ= 155°) is located on the loop 200

between strands β8 and β9, with His196 at its apex. 201

The model of the wild-type di-zinc structure includes 226 amino acid residues (41-312, 202

according to the BBL numbering (13)). The last serine residue at the C-terminus is missing. The 203

electron density was good enough to identify three zinc ions (with two in the active site and one 204

on the surface) and a sulphate ion in the active site. The composition of the visible solvent sphere 205

is mentioned in table 2. The Gly232–Phe236 mobile loop region had a low electron density and 206

the main chain for the residues was modelled in two conformations. The Phe236 side chain was 207


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not visible in the electron density map and other side chains were modelled with alternative 208

conformations. 209

CphA shows the typical αββα fold of the metallo-β-lactamase superfamily. The overall 210

fold is not altered by the presence of the second zinc. The di-zinc form has an average RMSD 211

value of 0.272 Å for backbone atoms with respect to the mono-zinc form. 212

The positions of the zinc ions were verified at the level of the electron density and by using 213

anomalous signals. Both zinc ions were refined to a 100 % occupancy and show an increased 214

mobility. This is indicated by the B-factors (17.68 and 20.63 for the first and the second zinc 215

ions, respectively) lying over the mean B-factor (9.81). The first zinc ion is found in the Asp120-216

Cys221-His263 site or “cysteine” site as previously observed in the mono-zinc form (14) (Figure 217

1). The distances between the zinc ion and the Asp120 carboxyl oxygen atom, the Cys221 218

sulphur atom, and the His263 side-chain nitrogen atom are 2.00, 2.26 and 2.09 Å, respectively. In 219

the mono-zinc form, these distances were 1.96, 2.20 and 2.05 Å, respectively (14). The 220

tetrahedral coordination sphere is completed by a sulphate ion from the crystallization solution at 221

a distance of 2.04 Å. 222

The second zinc ion is coordinated by the His118 and His196 residues. Both histidine 223

residues belong to the conserved “histidine” site in metallo-β-lactamases. The distances between 224

the zinc ion and the His118 ND1 and the His196 NE2 are 2.01 and 2.03 Å, respectively. In 225

CphA, the third ligand His116 is not conserved and is replaced by an asparagine residue, which is 226

not involved in the binding of the second zinc (the distance is more than 4 Å). A sulphate ion 227

(1.95 Å) and a water molecule (2.01Å) complete the vacant coordination positions of the zinc ion 228

in the “histidine” site, forming a tetrahedral coordination sphere. The sulphate ion acts as a 229

bridging agent between the zinc ions (Figure 1). The distances between the sulphate ion and zinc 230


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in the “cysteine” site and zinc in the “histidine” site are 2.04 and 1.95 Å, respectively. The 231

distance between both zinc ions is 4.08 Å. 232

The third observed zinc ion was found on the surface, away from the active site (about 17 233

Å). It is coordinated by His289 (2.09 Å from His NE) and the coordination sphere is completed 234

by three chloride ions (2.19, 2.25 and 3.17 Å). 235

236

Di-zinc form of the N220G mutant 237

The crystal structure of the di-zinc form of N220G was solved by molecular replacement 238

based on the mono-zinc structure (PDB code 1X8G). The structure was refined to 1.70 Å with 239

Rwork and Rfree values of 0.1308 of 0.1602, respectively. 240

The model of the N220G di-zinc structure included all the 227 residues of the protein (41-241

313 according to the BBL numbering (13)), three zinc ions (two in the active site and one on the 242

surface) and one sulphate ion located in the active site. The complete content of the solvent 243

sphere is described in table 2. The C-terminal residues were highly flexible. Ala195 was found in 244

a disallowed region of the Ramachandran plot. The electron density for the Gly232–Phe236 245

mobile loop region allowed a well-defined model to be constructed. 246

The active site of the N220G mutant with two bonded zinc ions had a conformation nearly 247

identical to that of the wild-type di-zinc enzyme. The RMSD for all main chain atoms was 0.216 248

Å. In contrast to the mono-zinc form of the N220G mutant where the zinc ion occupied two sites 249

1.5 Å apart (14), in the di-zinc form of the enzyme described here, the zinc ion was in the 250

classical “cysteine” site with distances to ligands identical to those in the wild-type structure. 251

Also, a sulphate ion and a water molecule (both with 50 % occupancy) were included in the 252

coordination sphere at distances of 2.04 and 2.27 Å, respectively. Under the new crystallisation 253


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conditions described above, a mono-zinc form of the N220G mutant was also obtained, in which 254

a fully occupied zinc ion was present in the canonical “cysteine” site (data not shown). 255

The second zinc ion could not be modelled with an occupancy higher than 0.8. This 256

second zinc ion shows a B-factor of 9.85, nearly the same as the mean B-factor (7.84) and in the 257

same range as the B-factor of the zinc ion in the “cysteine” site (10.50). This zinc ion was 258

coordinated by His118 and His196. The coordination sphere was completed by a water molecule 259

(2.02 Å) and the sulphate ion (2.15 Å). An additional water molecule was visible at a distance of 260

2.54 Å. 261

The third zinc ion was found nearly in the same position as in the wild-type structure. 262

Here, the zinc ion and the coordinating chloride ions exhibited 50 % occupancy. The coordination 263

sphere was affected by a water molecule and a disordered side chain from Glu292. 264

265

Comparison between the mono- and di-zinc structures of CphA 266

The binding of the second zinc ion in the active site has no influence on the global 267

structure of CphA. This is confirmed by the low RMSD values obtained by superposition of the 268

mono- and di-zinc forms. The differences between the mono- and di-zinc structures are limited to 269

the Gly232–Asn233 loop region (Figure 2). This loop, located at the entrance to the active site, is 270

known to be stabilized upon the binding of the substrate (14) or inhibitors (19, 22). For the wild-271

type di-zinc structure, this loop can exist in two conformations (both half occupied), 272

corresponding, respectively, to the open form (as in the mono-zinc structures 1X8G and 1X8H) 273

or the closed form (as in the structures with substrate or inhibitor in the active site, 1X8I, 2QDS 274

and 2GKL). In the di-zinc form of the N220G mutant, this loop is found in the closed form 275

(Figure 2). The closed conformation is stabilized by additional H-bonds between the Asn233 side 276

chain and the Ser235 side chain oxygen and backbone nitrogen. 277


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The active site of the di-zinc forms can be superimposed nearly perfectly over all the 278

previously available CphA structures (PDB codes 1X8G, 1X8H, 1X8I, 2QDS and 2GKL). For 279

example, the RMSD is 0.56 Å for the superposition of the “cysteine” and “histidine” sites over 280

their counterparts in 1X8G. The position of the zinc in the “cysteine” site is nearly identical, with 281

the exception of the mono-zinc N220G mutant where a disordered zinc ion was observed (14). 282

The coordinating His118 residue in the “histidine” site is not affected by the binding of the 283

second zinc. In contrast, the His196 residue displays a slight flexibility. It is noticeable that the 284

orientation of the imidazole ring of His196 in all X-ray structures is selected to achieve an 285

optimal pattern of contacts in the coordination sphere (H-bonds, zinc binding). Therefore, in di-286

zinc structures, the His196 imidazole ring is rotated by 180° compared to its position in the 287

mono-zinc structures (Figure 2). 288

289

Comparison with the active sites in B1 and B3 metallo-ββββ-lactamases 290

The active sites of all three metallo-β-lactamase subclasses show high degree of 291

correlation (RMSD values between 0.5 Å and 1.6 Å). In subclass B2, the coordination sphere of 292

the zinc in the “histidine” site is altered by the substitution of His116 by an asparagine residue. In 293

subclasses B1 and B3, the His116 residue is involved in the binding of the zinc ion in the 294

“histidine” site (Figure 3). Removing one coordinating residue causes this zinc ion to shift by 295

1.12, 1.03, 1.03 and 0.92 Å compared to BcII (PDB code 1BVT) (Figure 3), VIM-2 (PDB code 296

1KO3), VIM-2 (PDB code 1KO2) and L1 (PDB code 2FM6) structures, respectively. The 297

position of the zinc in the “cysteine” site is nearly unchanged when compared to the available 298

structures of subclass B1 enzymes. 299

300

Stability study 301


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As already reported (16), the helical contents of the apo-, mono- and di-zinc forms of the 302

wild-type CphA enzyme are similar. The apparent Tm values determined by CD spectroscopy 303

were 46.9, 58.7 and 61.6°C for the apo-, mono- and di-zinc forms, respectively. The mono-zinc 304

wild-type CphA is also less stable than the di-zinc form with urea as a denaturing reagent. 305

Transitions between native and denatured states occurred at 3.4 and 4.4 M urea for the mono- and 306

di-zinc forms, respectively. (data not shown) 307

The far-UV CD spectra of the apo-, mono- and di-zinc forms of the N220G mutant 308

exhibited small but significant differences (Figure 4a). However, as observed for the wild-type 309

enzyme, the helical content did not vary significantly. Thermal denaturation was irreversible in 310

all cases. The apparent Tm values determined by CD spectroscopy were 49.2, 61.7 and 62.9°C for 311

the apo-, mono- and di-zinc forms, respectively (Figure 4b). Also, the apparent Tm value 312

determined for the mono-zinc form of N220G using a thermal shift assay (Figure 4c) and by DSC 313

was 62°C but decreased to 50°C in the presence of 10 mM EDTA (Figure 4c). The mono-zinc 314

form of the N220G mutant was slightly less stable than the di-zinc form with urea as the 315

denaturing reagent. Transitions between native and denatured states occurred at 4.1 and 4.7 M 316

urea for the mono- and di-zinc forms, respectively (data not shown). 317

318

Is the “closed” position of the Gly232-Asn233 loop important for the inhibition by the second 319

zinc ion? - Analysis of the N233A mutant 320

An Asn233 residue is present in all metallo-β-lactamases with the exception of the L1, 321

BlaB and IND-1 enzymes (13). In CphA, the binding of a carbapenem substrate or inhibitor 322

modifies the Asn233 ψ angle so that its side chain closes the entrance of the active site in a 323

conformation stabilised by the formation of an H-bond between the oxygen of the Asn233 side-324


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chain and the hydroxyl of Ser235 (14, 19, 22). In the structure of the di-zinc form of CphA, the 325

loop adopts the closed position even in the absence of substrate, thus hindering access to the 326

active site (Figure 2). To explore the role of the Asn233 residue in the inhibition phenomenon, 327

the N233A mutant was expressed in Escherichia coli BL21 (DE3) pLysS Star, purified to 328

homogeneity and characterized. The mass of the protein was verified by electrospray ionisation 329

mass spectrometry (ESI-MS). Within experimental error, the mutant was found to exhibit the 330

expected mass (25144 Da measured vs. 25146 Da expected). The far UV CD spectrum of the 331

mutant enzyme showed the same α/β ratio as the wild type. The enzyme was stored in 15mM 332

sodium cacodylate (pH 6.5) with < 0.4 µM free zinc. Under these conditions, the N233A protein 333

contained one zinc ion per molecule as reported for the wild-type β-lactamase. The activity of the 334

mutant was measured in the absence of added Zn2+ (< 0.4 µM) with imipenem, nitrocefin and 335

CENTA (Table 3). The N233A mutant showed an increased Km value for imipenem. In contrast 336

to the wild-type enzyme, the N233A mutant was able to hydrolyse (albeit rather poorly) both 337

cephalosporins, with quite low Km values. The hydrolysis of imipenem by N233A was inhibited 338

by the binding of the second zinc ion, but the KD2 value decreased to 11 ± 2 µM compared to 46 339

µM for the wild-type enzyme. 340


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Discussion 341

There are three subclasses of metallo-β-lactamases (B1, B2 and B3), all of which require 342

Zn2+ for activity and can bind either one or two zinc ions (12, 13). Subclass B2 metallo-β-343

lactamases are active only in the mono-zinc form, because the binding of a second zinc ion 344

inhibits the enzyme in a non-competitive manner (16). This behaviour contrasts with that of B1 345

and B3 subclasses, where the presence of a second zinc ion in the active site has either little effect 346

or facilitates enzyme activity. Since it has thus far proven difficult to determine the structure of 347

the di-zinc form of a subclass B2 β-lactamase to investigate the basis of this inhibitory effect, we 348

set out to crystallise the di-zinc form of Aeromonas hydrophila CphA and solve its structure. 349

Previous attempts to determine the crystal structure of di-zinc A. hydrophila CphA have 350

not been successful, even with a high concentration of zinc in the mother liquor (14). It was 351

proposed that this might reflect the conditions used to grow crystals and/or the presence of a 352

carbonate ion in the active site. Also, it was suggested that the binding of the second zinc ion 353

required a change of active site conformation impossible to obtain in the crystal. Indeed, nuclear 354

magnetic resonance (NMR) measurements indicate major conformation changes upon binding of 355

the second zinc ion (C. Damblon, personal communication). However, CD spectra showed that 356

the secondary structures of the mono-zinc forms of the wild-type (16) and N220G CphA enzymes 357

are only slightly different from those of the di-zinc forms. By modifying the crystallization 358

conditions, we succeeded in obtaining CphA crystals with two zinc ions in the active site. 359

Moreover, the overall αββα conformation of the enzyme was not affected by the presence of a 360

second zinc ion. The di-zinc form of CphA was obtained by soaking the crystal into a solution 361

containing a zinc concentration well above the KD2 value. In the crystal, major movements 362

leading to the significant unfolding of the protein as observed by NMR are impossible. 363


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In the mono-zinc form, a carbonate ion was present in the active site (14). In our 364

structures, a sulphate ion from the crystallization solution was bound to both zinc ions. 365

As previously postulated (4, 34), the second inhibitory zinc ion is located in a slightly 366

modified “histidine” binding site with the two conserved His118 and His196 residues as ligands. 367

The Met146 residue, proposed as one of the inhibitory zinc ion ligands together with His118 in 368

the ImiS metallo-β-lactamase (6), was 12.54 Å from this ion in CphA and the sulphur atom points 369

in the opposite direction. Also, the space between the His118 and Met146 residues is occupied by 370

the backbone of the Asn114-His118 loop, connecting strand β6 and helix α2, and thus does not 371

allow the accommodation of a zinc ion. The sequence of ImiS is 96% identical to that of CphA, 372

so it would be remarkable if these few substitutions produced such a radically different 373

mechanism for the binding of the inhibitory zinc ion. 374

This “atypical” coordination sphere with only two of the three conserved histidine 375

residues (figure 3) could probably explain the rather high value of the dissociation constant for 376

the “histidine” site in CphA (46 µM) compared to that in subclasses B1 and B3. The value of the 377

dissociation constant for the “histidine” site is 1.8 nM for BcII (a subclass B1 enzyme) and 378

subclass B3 enzymes have high affinity constants for both binding sites (KD < 6 nM) (35). 379

Vanhove et al. proposed an explanation for the inhibition of subclass B2 enzymes by the 380

second zinc ion. The side-chain of the Cys221 which is essential for binding the first zinc ion 381

would be displaced by the binding of the second zinc ion (34). However, a comparison between 382

mono- and di-zinc structures showed that this residue was not displaced. On the basis of the 383

results obtained here, we can confirm that the binding of the inhibitory zinc ion to the 384

catalytically important His118 and His196 residues prevents them from playing their catalytic 385

roles in the hydrolysis of carbapenems (4, 14) where His118 is the general base which activates 386


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the hydrolytic water molecule and His196 contributes to the oxyanion hole by forming a H-bond 387

with the carbonyl oxygen of the β-lactam bond (14). This model is supported by the very weak 388

activities of the H118A and H196A mutants (4). This conclusion reinforces the hypothesis that 389

the nucleophile is not adequately metal activated in B2 β-lactamases (30, 36). Superimposition of 390

the structures of the dizinc forms of BcII (B1) and CphA shows that in the latter, the water 391

molecule does not bridge the two zinc ions but is only in interaction with that in the “histidine” 392

site (figure 5). It is possible that an interaction with this sole zinc ion does not sufficiently 393

decrease the water pKa so that the concentration of OH- ions remains very low. Moreover, 394

His118 which now serves as a zinc ligand can non longer play the role of general base. The 395

closure of the Gly232–Asn233 loop in the di-zinc form could also help to inhibit the enzyme, but 396

Asn233 probably plays only a minor role since the N233A mutant is still inhibited by the binding 397

of the second zinc ion. Finally, a third zinc ion is bound to a superficial histidine residue, but this 398

is unlikely to be involved in the inhibition phenomenon. Its binding is probably due to the high 399

zinc concentration utilized. 400

Whereas the N233S mutant of ImiS exhibits kinetic parameters similar to those of wild-401

type enzyme (30), the N233A mutant of CphA shows an increased Km value for imipenem, and 402

thus seems to play a role in the binding of this substrate. Two-thirds of all sequenced metallo-β-403

lactamases have an Asn at position 233 (12, 13), and this residue was shown to be involved in 404

substrate binding and activation by interacting electrostatically with the substrate β-lactam 405

carbonyl (37). A same role for Asn233 residue in substrate binding by CphA was already 406

predicted by computational modelling (36) and is in agreement with our results. 407

In contrast to the zinc ion located in the “cysteine” site, the second zinc ion does not play 408

an important structural role. Indeed, the binding of the first zinc ion stabilizes the wild-type and 409


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N220G CphA enzymes significantly, whereas the second zinc ion has a much more limited 410

impact on stability. Both the mono- and di-zinc forms of the N220G mutant are somewhat more 411

stable than their wild-type counterparts, perhaps explaining why crystals of the N220G mutant 412

are obtained more easily than those of the wild-type enzyme. 413

In conclusion, the catalytic metal ion is located in the “cysteine” site of CphA and the 414

second inhibitory zinc ion binds to a slightly modified “histidine” site. The “histidine” site has 415

first been considered as the catalytic site in the mononuclear metallo-β-lactamases. Indeed, in the 416

first reported monozinc structures (B1 enzymes such as BcII, VIM-2, VIM-4 and SPM-1), the 417

single metal ion was shown to be located in the “histidine” site (5, 15, 25). In contrast, a B3 418

enzyme called GOB was shown to be active as a monozinc enzyme with the metal ion located in 419

the “cysteine” site (24). Recently, Vila and co-workers proposed that B1 enzymes can be active 420

in their mono- and dizinc forms and that in mono-cobalt BcII the metal ion is localized in the 421

“cysteine” site (23, 31). Two mononuclear mutants of BcII in which each of the metal binding 422

sites was selectively removed produced inactive variants. The authors concluded that the 423

mononuclear form can be active only if assisted by residues at the other binding site (1). 424

According to the model proposed by Tioni et al. (31), the metal ion in the “histidine” site would 425

activate the hydrolytic water molecule in the dizinc enzyme. Vila and co-workers proposed that 426

this is facilitated by a net of H-bond interactions in the mononuclear enzyme (23, 31) probably 427

including His118 or Asp120, as already proposed for CphA (4, 14, 36). However, Asp120 428

appears unlikely because it already coordinates a zinc ion. In subclass B2, as previously 429

postulated by Vanhove et al. (34), Asn116 is not involved in the binding of the second metal ion 430

but the N116H (34) and N116H-N220G (2) mutants could have a reconstituted His116-His118-431

His196 site and behave similarly to B1 enzymes. Indeed, the N116H-N220G mutant has an 432

extended substrate spectrum and its di-zinc form is active (2). 433


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The crystal structure of the subclass B2 CphA β-lactamase in its metal-inhibited form 434

provides thus an answer to the structural characterization of the inhibitory site that has been 435

elusive and controversial so far. 436


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Acknowledgements 437

The work in Liège was supported by the Belgian Federal Government (PAI P5/33) and 438

grants from the FNRS (Brussels, Belgium, FRFC grant n° 2.4511.06 and Lot. Nat. 9.4538.03). 439

C.B. is a FRS/FNRS post-doctoral researcher. 440


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References 441

1. Abriata, L. A., L. J Gonzalez, L. I. Llarrull, P. E. Tomatis, W. K. Myers, A. L. Costello, 442

D. L Tierney, and A. J. Vila. 2008. Engineered mononuclear variants in Bacillus cereus 443

metallo-β-lactamase are inactive. Biochemistry, 47:8590–8599. 444

2. Bebrone, C., C. Anne, K. De Vriendt, B. Devresse, J. Van Beeumen, J.M. Frère, and M. 445

Galleni. 2005. Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA 446

metallo-β-lactamase by site-directed mutagenesis, J. Biol. Chem.,17:180-188. 447

3. Bebrone, C. 2007. Metallo-β-lactamases (classification, activity, genetic organization, 448

structure, zinc coordination) and their Superfamily. Biochem. Pharma. 74:1686-1701. 449

4. Bebrone, C., C. Anne, F. Kerff, G. Garau, K. De Vriendt, R. Lantin, B. Devreese, J. Van 450

Beeumen, O. Dideberg, J.M. Frère, and M. Galleni. 2008. Mutational analysis of the zinc and 451

substrate binding sites in the CphA metallo-β-lactamase from Aeromonas hydrophila, Biochem. 452

J, 114:151-159. 453

5. Carfi, A., S. Parès, E. Duée, M. Galleni, C. Duez, J.M. Frère, and O. Dideberg. 1995. The 454

3-D structure of a zinc metallo-β-lactamase from Bacillus cereus reveals a new type of protein 455

fold. EMBO J. 14:4914-4921. 456

6. Costello, A.L., N.P. Sharma, K.W. Yang, M.W. Crowder, and D.L. Tierney. 2006. X-ray 457

absorption spectroscopy of the zinc-binding sites in the class B2 metallo-β-lactamase ImiS from 458

Aeromonas veronii bv. sobria. Biochemistry. 45:13650-13658. 459

7. Crawford, P.A., K.W. Yang, N. Sharma, B. Bennett, and M.W. Crowder. 2005. 460

Spectroscopic studies on cobalt(II)-substituted metallo-β-lactamase ImiS from Aeromonas 461

veronii bv. sobria. Biochemistry. 44:5168-5176. 462


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

24

8. Daiyasu, H., K. Osaka, Y. Ishino, and H. Toh. 2001. Expansion of the zinc metallo-463

hydrolase family of the β-lactamase fold. FEBS Lett. 503:1-6. 464

9. Emsley, P., and K.Cowtan. 2004. Coot: model-building tools for molecular graphics. Acta 465

Crystallogr. D60:2126-2132. 466

10. Felici, A., G. Amicosante, A. Oratore, R. Strom, P. Ledent, B. Joris, L. Fanuel, and J.M. 467

Frère. 1993. An overview of the kinetic parameters of class B β-lactamases. Biochem.J. 468

291:151-155. 469

11. Fonseca, F., A. Correia, and J. Spencer. 2008. Structural and kinetic characterisation of 470

Sfh-1, the B2 metallo-β-lactamase of Serratia fonticola. p.46. Proceedings of the 10th β-471

lactamase meeting, Eretria, Greece. 472

12. Galleni, M., J. Lamotte-Brasseur, G.M. Rossolini, J. Spencer, O. Dideberg, J.M. Frère, 473

and the Metallo-ββββ-lactamases Working Group. 2001. Standard numbering scheme for class B 474

β-lactamases. Antimicrob.Agents Chemother., 45:660-663. 475

13. Garau, G., I. Garcia-Saez, C. Bebrone, C. Anne, P.S. Mercuri, M. Galleni, J.M. Frère, 476

and O. Dideberg. 2004. Update of the standard numbering scheme for class B β-lactamases. 477

Antimicrob.Agents Chemother., 48:2347-2349. 478

14. Garau, G., C. Bebrone, C. Anne, M. Galleni, J.M. Frère, and O. Dideberg. 2005. A 479

Metallo-β-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase 480

CphA and its Complex with Biapenem. J. Mol. Biol. 345:785-795. 481

15. Garcia-Saez, I., J.D. Docquier, G.M. Rossolini, and O. Dideberg. 2008. The three-482

dimensional structure of VIM-2, a Zn-β-lactamase from Pseudomonas aeruginosa in its reduced 483

and oxidised form. J. Mol. Biol. 375:604-611. 484


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

25

16. Hernandez-Valladares, M., A. Felici., G. Weber, H.W. Adolph, M. Zeppezauer, G.M. 485

Rossolini, G. Amicosante, J.M. Frère, and M. Galleni. 1997 Zn(II) dependence of the 486

Aeromonas hydrophila AE036 metallo-β-lactamase activity and stability. Biochemistry. 487

36:11534-11541. 488

17. Hernandez-Valladares, M., M. Kiefer, U. Heinz, R. Paul Soto, W. Meyer-Klaucke, H. 489

Friederich Nolting, M. Zeppezauer, M. Galleni, J.M. Frère, G.M. Rossolini, G. Amicosante 490

and H.W. Adolph. 2000. Kinetic and spectroscopic characterization of native and metal-491

substituted β-lactamase from Aeromonas hydrophila AE036. FEBS Lett. 467:221-225. 492

18. Hooft, R.W.W., G. Vriend, C. Sander, E.E. and Abola. 1996. Errors in protein structures. 493

Nature. 381:272-272. 494

19. Horsfall, L.E., G. Garau, B.M. Liénard, O. Dideberg, C.J. Schofield, J.M. Frère, and M. 495

Galleni. 2007 Competitive inhibitors of the CphA metallo-β-lactamase from Aeromonas 496

hydrophila. Antimicrob. Agents Chemother. 51:2136-2142. 497

20. Kabsch, W. 1993. Automatic processing of rotation diffraction data from crystals of initially 498

unknown symmetry and cell constants. J. Appl. Cryst. 26:795-800. 499

21. Laskowski, R. A., M.W. MacArthur, D.S. Moss, and J.M. Thornton. 1993. PROCHECK: 500

a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26:283-291. 501

22. Liénard, B.M.R., G. Garau, L. Horsfall, A.I. Karsisiotis, C. Damblon, P. Lassaux, C. 502

Papamicael, G.C.K. Roberts, M. Galleni, O. Dideberg, J.M. Frère, and C.J. Schofield. 2008. 503

Structural basis for the broad-spectrum inhibition of metallo-β-lactamases by thiols. Org. Biomol. 504

Chem. 6:2282-2294. 505

23. Llarull, L.I., M.F. Tioni and A.J. Vila. 2008. Metal content and localization during turnover 506

in Bacillus cereus metallo-β-lactamase. J. Am. Chem. Soc. 130:15842-15851. 507


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

26

24. Morán-Barrio, J., J.M. González, M.N. Lisa, A.L. Costello, M. Dal Peraro, P. Carloni, 508

B. Bennett, D.L. Tierney, A.S. Limansky, A.M. Viale, and A.J. Vila. 2007. The metallo-β-509

lactamase GOB is a mono-Zn(II) enzyme with a novel active site. J. Biol. Chem. 282:18286-510

18293. 511

25. Murphy, T.A., L.E. Catto, S.E. Halford, A.T. Hadfield, W. Minor, T.R. Walsh, and J. 512

Spencer. 2006. Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into 513

variable zinc affinity of metallo-ββββ-lactamases. J.Mol.Biol. 357:890-903. 514

26. Murshudov, G.N., A.A. Vagin, and E.J. Dodson. 1997. Refinement of macromolecular 515

structures by the maximum-likelihood method. Acta Crystallogr. D53:240-255. 516

27. Painter, J., E.A. and Merritt. 2006. Optimal description of a protein structure in terms of 517

multiple groups undergoing TLS motion. Acta Crystallogr. D62:439-450. 518

28. Perrakis, A., R. Morris, and V.S. Lamzin. 1999. Automated protein model building 519

combined with iterative structure refinement. Nat Struct Biol. 6(5):458-463. 520

29. Segatore, B., O. Massida, G. Satta, D. Setacci, and G. Amicosante. 1993. High specificity 521

of cphA-encoded metallo-β-lactamase from Aeromonas hydrophila AE036 for carbapenems and 522

its contribution to β-lactam resistance. Antimicrobial Agents and Chemother. 37:1324-1328. 523

30. Sharma, N.P., C. Hajdin, S. Chandrasekar, B. Bennett, K.W. Yang, and M.W. Crowder. 524

2006. Mechanistic studies on the mononuclear ZnII-containing metallo-β-lactamase ImiS from 525

Aeromonas sobria. Biochemistry. 45:10729-10738. 526

31. Tioni, M.F., L.I. Llarull, A.A. Poeylaut-Palena, M.A. Marti, M. Saggu, G.R. Periyannan, 527

E.G. Mata, B. Bennett, D.H. Murgida, and A.J. Vila. 2008. Trapping and characterization of a 528

reaction intermediate in carbapenem hydrolysis by Bacillus cereus metallo-β-lactamase. J. Am. 529

Chem. Soc. 130:15852-15863. 530


http://aac.asm.org/

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http://aac.asm.org/

27

32. The CCP4. 1994. CCP4 suite: programs for protein crystallography. Acta Crystallog. Sect. 531

D. 50:760-763. 532

33. Ullah, J.H., T.R. Walsh, I.A. Taylor, D.C. Emery, C.S. Verma, S.J. Gamblin, and J. 533

Spencer. 1998. The crystal structure of the L1 metallo-β-lactamase from Stenotrophomonas 534

maltophilia at 1.7 Å resolution. J.Mol.Biol. 284:125-136. 535

34. Vanhove, M., M. Zakhem, B. Devreese, N. Franceschini, C. Anne, C. Bebrone, G. 536

Amicosante, G.M. Rossolini, J. Van Beeumen., J.M. Frère, and M. Galleni. 2003. Role of 537

Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-β-lactamase. 538

Cell Mol Life Sci. 60:2501-2509. 539

35. Wommer, S., S. Rival, U. Heinz, M. Galleni, J.M. Frère, N. Franceschini, G. 540

Amicosante, B; Rasmussen, R. Bauer, and H.W. Adolph. 2002. Substrate-activated zinc 541

binding of metallo-β -lactamases: physiological importance of mononuclear enzymes. J. Biol. 542

Chem. 277:24142-24147. 543

36. Xu, D., D. Xie, and H. Guo. 2006. Catalytic mechanism of class B2 metallo-β-lactamase. J. 544

Biol. Chem. 281:8740-8747. 545

37. Yanchak, M.P., Taylor, R.A. and M.W. Crowder. 2000. Mutational analysis of metallo-β-546

lactamase CcrA from Bacteroides fragilis. Biochemistry. 39:11330-11339. 547


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Table 1. Composition of the two metal-binding sites in the three metallo-ββββ-lactamase 548

subclasses. * This “histidine” site is still putative, since the 3D structure of the GOB enzyme is 549

not available. 550

551

Enzymes "Histidine" site "Cysteine" site

B1 MβLs His116-His118-His196 Asp120-Cys221-His263

B2 MβLs His118-His196 Asp120-Cys221-His263

B3 MβLs His116-His118-His196 Asp120-His121-His263

B3 MβL GOB (Gln116)-His118-His196* Asp120-His121-His263


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Table 2. X-ray data collection and structure refinement 552

553

CphA WT di-zinc CphA N220G di-zinc A. Data Collection statistics Resolution range (Å) 19.7-2.03 19.6-1.70 Wavelength (Å) 1.5418 1.5418 Unit cell parameters Space group C2221 C2221 a (Å) 42.80 42.68 b (Å) 101.05 101.06 c (Å) 116.49 116.83 Rsym 0.062 0.032 No. observed reflections 235065 206898 No. unique reflections 16754 28046 Completeness (%) 99.7 99.7 I/sigma(I) 34.4 42.1 Multiplicity 14 7.4 B (Wilson) 15.69 11.96 B. Refinement statistics No. reflections used 16754 28046 Molecules/asymmetric unit 1 1 Rworking 0.1568 0.1388 Rfree 0.2020 0.16024 Residues in disallowed region 1 1 RMS deviation from ideal Bonds (Å) 0.011 0.011 Angles (deg.) 1.217 1.550 Mean B factor 9.81 7.84 No. atoms (non-H) 2090 2198 No. atoms (protein) 1850 1882 No. of H20 molecules 202 252 Other molecules/ions Zn2+ SO4 CO3

Cl- Glycerol

3 4 2 7 -

3 7 - 7 3


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Table 3. Kinetic parameters of the wild-type and N233A CphA enzymes. All the 554

experiments were performed at 30°C in 15 mM cacodylate (pH 6.5), [Zn2+] < 0.4 µM. Standard 555

errors were below 10%. NH: no observed hydrolysis 556

557

WT N233A

kcat (s-1)

Km (µM)

kcat/Km (M-1s-1)

kcat (s-1)

Km (µM)

kcat/Km (M-1s-1)

Imipenem 1200 340 3 500 000 > 700 > 1000 700 000

Nitrocefin 0.008 1300 6 0.03 55 520

CENTA NH NH NH 0.0035 14 250


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Figure legends 558

559

Figure 1 560

Structure of the wild-type CphA metal-binding sites. Zinc ions and water molecule are shown 561

as grey and red spheres, respectively. ZnHis is the zinc ion that binds in the “histidine” site while 562

ZnCys is the zinc ion that binds in the “cysteine” site. The anomalous map at a contour level of 4 σ 563

is shown in orange. Relevant distances between zinc ions and ligands are indicated. 564

565

Figure 2 566

Superposition of the mono- (in grey) and di-zinc (in yellow) forms of the N220G CphA 567

enzyme. The Gly232–Asn233 loop region of the di-zinc form is represented in red to underline 568

the conformational change. The positions of the His196 residue in the mono-form (H196mono) and 569

in the di-zinc form (H196di) are represented. The zinc ions in the “histidine” site (ZnHis) and in the 570

“cysteine” site (ZnCys) are shown as grey spheres. The distance between His196di and the zinc ion 571

located in the “histidine” site is indicated. 572

573

Figure 3 574

The “histidine” site in CphA (a) and in BcII (PDB code 1BVT) (b). Zinc ions and water 575

molecules are represented as grey and red spheres, respectively. 576

577

Figure 4 578

Structural studies of the N220G mutant 579


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a) Circular dichroism spectra of the apo-enzyme (dotted line), mono-zinc (dashed line) and 580

di-zinc forms (solid line) of the N220G enzyme (0.3 mg/ml in both cases). 581

b) Thermal denaturation of the N220G mutant (0.5 mg/ml): fraction of native protein vs 582

temperature (°C). Apo-N220G (dotted line), mono-zinc N220G (dashed line), di-zinc 583

N220G (solid line). 584

c) Thermal shift assay for N220G (0.3 mg/ml). N220G + 10 mM EDTA (dotted line), mono-585

zinc N220G (dashed line). 586

587

Figure 5 588

Superimposition of the structures of the di-zinc forms of BcII (PDB code 2BFK) and CphA. 589

Zinc ligands of BcII and CphA are represented as grey and green sticks, respectively. Zinc ions of 590

BcII and CphA are shown as grey and green spheres, respectively. ZnHis is the zinc ion that binds 591

in the “histidine” site while ZnCys is the zinc ion that binds in the “cysteine” site. Water molecules 592

of BcII and CphA are shown as purple and red spheres, respectively. The sulphate ion that 593

bridges the two zinc ions in the CphA structure (SO4CphA) is also represented, as well as glycerol, 594

the fifth ligand of ZnCys in BcII (glycerolBcII). 595

596

597


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The structure of the di-zinc subclass B2 metallo- bbbb...

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