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1 The structure of the di-zinc subclass B2 metallo--lactamase CphA reveals 2 that the second inhibitory zinc ion binds in the “histidine” site. 3 4 Carine Bebrone1,3,*,§, Heinrich Delbrück2,*, Michaël B. Kupper2, Philipp Schlömer2, 5 Charlotte Willmann2, Jean-Marie Frère1, Rainer Fischer2, Moreno Galleni3 and Kurt M. V. 6 Hoffmann2 7 8 1 9 Liège, Belgium Centre for Protein Engineering, University of Liège, Allée du 6 Août B6, Sart-Tilman 4000 10 2 11 Forckenbeckstrasse 6, 52074 Aachen, Germany 12 3 13 Belgium Institute of Molecular Biotechnology, RWTH-Aachen University, c/o Fraunhofer IME, Biological macromolecules, University of Liège, Allée du 6 Août B6, Sart-Tilman, 4000 Liège, 14 15 * both authors contributed equally to this work 16 17 Running title: The inhibitory zinc ion binds in the “histidine” site of CphA 18 19 20 § 21 Allée du 6 Août B6, Sart-Tilman, 4000 Liège, Belgium, Tel.: 003243663315, Fax: 22 003243663364, e-mail: [email protected] Corresponding author: Carine Bebrone, Centre for Protein Engineering, University of Liège, 23 1 24 Abstract 25 Bacteria can defend themselves against -lactam antibiotics through the expression of 26 class B -lactamases, which cleave the -lactam amide bond and render the molecule harmless. 27 There are three subclasses of class B -lactamases (B1, B2 and B3), all of which require Zn2+ for 28 activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo--lactamases 29 are most active as di-zinc enzymes, subclass B2 enzymes such as Aeromonas hydrophila CphA 30 are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to 31 determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals 32 allowed us to solve the crystal structures of the di-zinc forms of the wild-type enzyme and 33 N220G mutant. The first zinc ion binds in the “cysteine” site, as previously determined for the 34 mono-zinc form of the enzyme. The second zinc ion occupies a slightly modified “histidine” site, 35 where the conserved His118 and His196 residues act as metal ligands. This atypical coordination 36 sphere probably explains the rather high dissociation constant for the second zinc ion compared 37 to those observed in enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results 38 from immobilization of the catalytically-important His118 and His196 residues, as well as the 39 folding of the Gly232–Asn233 loop into a position that covers the active site. 40 41 42 2 43 Introduction 44 Class B -lactamases (also called zinc -lactamases or metallo--lactamases) play a key 45 role in bacterial resistance to -lactam antibiotics by efficiently catalyzing the hydrolysis of the 46 -lactam amide bond. The existence of such enzymes is a particular concern because they are 47 effective against most -lactam antibiotics (including the carbapenems), the corresponding genes 48 are easily transferred between bacteria and there are no clinically useful inhibitors. On the basis 49 of the known sequences, three different lineages, identified as subclasses B1, B2, and B3, can be 50 characterized (12, 13). All class B -lactamases possess two potential zinc-binding sites and 51 share a small number of conserved motifs bearing some of the residues that coordinate the zinc 52 ion(s), notably His/Asn/Gln116-Xaa-His118-Xaa-Asp120 and Gly/Ala195-His196-Ser/Thr197 53 (13). Structural analysis of subclass B1 enzymes shows that one zinc ion has a tetrahedral 54 coordination sphere involving His116, His118, His196 and a water molecule or OH- ion 55 (“histidine” site), whereas the other has a trigonal-pyramidal coordination sphere involving 56 Asp120, Cys221, His263 and two water molecules (“cysteine” site) (Table 1). One 57 water/hydroxide serves as a ligand for both metal ions (3). In the mononuclear structures of B1 58 enzymes (BcII, VIM-2, SPM-1 and VIM-4), the sole metal ion was found to be located in the 59 “histidine” site (5, 15, 25 and P. Lassaux, unpublished data). In some monozinc B1 structures, the 60 Cys221 residue is found under an oxidized form. In subclass B3, the “histidine” site is similar to 61 that found in subclass B1, but Cys221 is replaced by a serine residue and the second zinc ion is 62 coordinated by Asp120, His121, His263 and the nucleophilic water molecule which again forms 63 a bridge between the two metal ions (Table 1) (33). 64 Aeromonas hydrophila CphA is a subclass B2 metallo--lactamase characterized by a 65 uniquely narrow specificity. CphA efficiently hydrolyses only carbapenems and shows very poor 3 66 activity against penicillins and cephalosporins, in contrast to subclass B1 and B3 enzymes which 67 hydrolyse nearly all -lactam compounds, with the exception of monobactams (10, 29). In 68 contrast to subclass B1 and B3 enzymes which are more active as di-zinc species, subclass B2 is 69 inhibited in a non-competitive manner by the binding of a second zinc ion. For CphA, the 70 dissociation constant of the second zinc ion (KD2) is 46 µM at pH 6.5 (16). In agreement with 71 EXAFS studies (17) and site-directed mutagenesis (34), the crystallographic structure of CphA 72 shows that the first zinc ion is in the “cysteine” site (14). However, the second binding site in 73 subclass B2 enzymes remains to be determined because Garau and colleagues could not produce 74 crystals of the di-zinc form despite presence of zinc concentration well above KD2 (14). The 75 structure of Sfh-1, a subclass B2 enzyme from Serratia fonticola, has been solved recently, once 76 again with only one zinc ion in the active site (11). In subclass B2, the His116 residue found 77 throughout the metallo--lactamase superfamily (with the exception of the B3 GOB enzymes 78 where Gln is present) is replaced by an asparagine (8, 12, 13). It has been previously shown that 79 the Asn116 residue has no role in the binding of the zinc ions in CphA (34). On the basis of 80 spectroscopic studies with the Aeromonas veronii cobalt-substituted ImiS enzyme, Crawford and 81 co-workers postulated that the second metal binding site was not the traditional “histidine” site 82 but a site, remote from the active site, which involves both His118 and Met146 as zinc ligands (6, 83 7). However, a recent study of potential zinc ligand mutants contradicts this hypothesis and 84 indicates that the position of the second zinc ion in CphA is probably equivalent to the “histidine” 85 site observed in subclasses B1 and B3 enzymes, with His118 and His 196 involved in the binding 86 of this second zinc perhaps with Cys221 (or Asn116 or Asp120) as the third ligand (4). 87 Since crystals of the wild-type CphA protein could not be obtained directly, single-site 88 mutants were engineered by site-directed mutagenesis and overproduced. These mutants were 4 89 selected in order to introduce residues that are conserved in either subclass B1 or B3, or both (2). 90 Among these mutants, the N220G mutant (2) easily yielded crystals which were used as seeds to 91 grow wild-type CphA crystals (14). The kinetic parameters of the wild-type and N220G mutant 92 CphA enzymes were similar, indicating strong conservation of enzymatic properties in the mutant 93 (2). However, the N220G mutation results in a slightly higher KD2 value (86 µM) (2). We 94 therefore set out to solve the crystallographic structures of the di-zinc forms of the wild-type 95 CphA enzyme and its N220G mutant. We confirmed that the second zinc ion occupies a slightly 96 modified “histidine” site, where the conserved His118 and His196 residues act as metal ligands. 97 The implication of this discovery on what is known about the coordination of zinc ions by 98 metallo--lactamases is discussed. We propose an explanation for the inhibition by the second 99 zinc ion. Moreover, the role of the Asn233 residue in this inhibition phenomenon is also 100 apprehended based on a site-directed mutagenesis study. In this work, a structural role for the 101 zinc ions is also investigated. 102 5 103 Materials and Methods 104 Protein purification and crystallization 105 Site-directed mutagenesis, protein expression and purification of the proteins were carried 106 out as described previously (2), with the addition of a third purification step consisting of a size 107 exclusion chromatography (Hiload 16/60 Superdex 75 prep grade column, Pharmacia Biotech). 108 The purified enzyme solution was dialyzed against 15 mM sodium cacodylate (pH 6.5). 109 Monodispersity of the protein solutions and the hydrodynamic radii of the dissolved protein 110 molecules were checked by Dynamic Light Scattering (DLS). The crystallisation conditions used 111 previously (14) were not easy to reproduce because the crystallisation drop comprised two 112 phases. Therefore, a screen was carried out to find new conditions. Initial screens were carried 113 out at 8°C using commercial Hampton Crystal Screens 1 and 2, Grid Screen Ammonium 114 Sulphate and Grid Screen PEG 6000 (Hampton Research, California, USA). N220G CphA (10 115 mg/ml) was crystallized from 2.2–2.4 M ammonium sulphate and 0.1 M MES (pH 6.0–6.5), 116 using the sitting drop method with new crystallisation plates designed by Taorad GmbH (Aachen, 117 Germany). The (1 µl) reservoir solution was mixed with the protein solution (1 µl) and the 118 mixture was left to equilibrate against the solution reservoir. Typically, orthorhombic crystals of 119 the N220G mutant grew within a few days to dimensions of 80 x 100 x 100 µm and belong to 120 space group C2221 (a=42.68, b=101.06 and c=116.82). Crystals of the wild-type CphA enzyme 121 were obtained under similar conditions using mutant micro-crystals as starting seeds and showed 122 similar orthorhombic symmetry in space group C2221 (a=42.80, b=101.50 and c=116.49). Before 123 data collection, 1µl of 100 mM ZnCl2 was added to the drops containing the wild-type and the 124 mutant crystals and soaked for one day. 125 6 126 Data collection and processing 127 For data collection, wild-type and mutant crystals were transferred to a cryoprotectant 128 solution containing reservoir solution supplemented with 30% (v/v) glycerol. The mounted 129 crystals were flash-frozen in a liquid nitrogen stream. Near-complete X-ray data sets were 130 collected using a Bruker FR591 rotating anode X-ray generator and a Mar345dtb detector. 131 Diffraction data were processed with XDS (20) and scaled with SCALA from the CCP4-Suite 132 (32). 133 134 Structure determination and refinement 135 The structures of di-zinc variants from the wild-type and N220G mutant enzymes were 136 solved using a molecular replacement approach with Molrep (32), and the mono-zinc structure of 137 CphA (PDB file 1X8G) as starting model. The resulting model was rebuilt with ARP/wARP (28). 138 The structures were refined in a cyclic process including manual inspection of the electron 139 density with Coot (9) and refinement with Refmac (26). The refinement to convergence was 140 carried out with isotropic B-values and using TLS parameter (27). The positions of the zinc, 141 sulphate and chloride ions were verified by calculating anomalous maps and the occupancies of 142 the ions were determined by disappearing of difference electron density. Alternative 143 conformations were modelled for a number of side chains and occupancies were adjusted to yield 144 similar B-values for the disordered conformations. 145 146 Circular dichroism (CD) spectroscopy 147 Circular dichroism (CD) spectra for the apo-, mono-and di-zinc forms of the wild-type 148 and N220G enzymes (0.3 mg/ml) were obtained using a JASCO J-810 spectropolarimeter. The 7 149 apoenzymes were obtained in the presence of 10 mM EDTA. The di-zinc forms were obtained in 150 the presence of 500 µM Zn2+. The spectra were scanned at 25°C with 1 nm steps from 200 to 250 151 nm (far UV). Thermal stability was assessed for the apo-, mono- and di-zinc forms of the wild- 152 type and N220G proteins using CD spectroscopy at 220 nm with increasing temperature (25°C– 153 85°C). Urea denaturation studies of the wild-type and N220G enzymes were also carried out in 154 the presence and absence of 500 µM Zn2+. The mono- and di-zinc enzymes were incubated for 155 18h in increasing concentrations of urea (0–8 M) at 4°C. Stability was assessed for the mono- and 156 di-zinc forms of both proteins using CD spectroscopy at 220 nm with increasing concentrations 157 of urea. 158 159 Thermal shift assay 160 Thermal shift assays were carried out using a LightCycler real-time PCR instruments 161 (Roche Diagnostics). Briefly, 10 µl of wild-type CphA enzyme or the N220G mutant (0.3 mg/ml) 162 was mixed with 10 µl of 5000 x Sypro Orange (Molecular Probes) diluted 1:500 in 15 mM 163 cacodylate (pH 6.5). Samples were heat-denatured from 25 to 90°C at a rate of 0.5°C per minute. 164 Protein thermal unfolding curves were monitored by detecting changes in Sypro Orange 165 fluorescence. The inflection point of the fluorescence vs temperature curves was identified by 166 plotting the first derivative over the temperature and the minima were referred to as the melting 167 temperature (Tm). Buffer fluorescence was used as the control. 168 169 Differential Scanning Calorimetry (DSC) 170 Thermal denaturation was investigated using a NDSC 6100 differential scanning 171 calorimeter (Calorimetry Sciences Corp., Lindon, USA) after dialysis of the N220G enzyme 8 172 sample (1 mg/ml) against 15 mM cacodylate (pH 6.5). The dialysis buffer was used as reference. 173 The data were collected and analysed with a MCS Observer software package assuming a two- 174 state folding mechanism. 175 176 Analysis of the N233A mutant 177 The Quik Change Site-directed mutagenesis kit (Stratagene Inc., La Jolla, Calif.) was used 178 to introduce the N233A mutation into CphA, using pET9a-CphA WT as the template, forward 179 primer 180 CGGCAAAGCTCAGGGCGCCCAGCTTCTCC-3′. The vector was then introduced into E. coli 181 strain BL21 (DE3) pLysS Star. Overexpression and purification of the mutant protein, CD 182 spectroscopy, electrospray ionization-mass spectrometry (ESI-MS) and the determination of 183 metal content, kinetic parameters and residual activity in the presence of increasing 184 concentrations of zinc were carried out as previously described (2, 4, 34). 5′-GGAGAAGCTGGGCGCCCTGAGCTTTGCCG-3′ and reverse primer 5′- 185 186 187 188 Protein Data Bank accession codes Coordinates and structure factors were deposited in the Protein Data Bank using accession codes 3F90 (di- zinc form of wild-type CphA) and 3FAI (di-zinc form of the N220G mutant). 189 9 190 Results 191 Structure determination. 192 The crystal structures of the zinc saturated wild-type and N220G CphA enzymes were 193 solved. Stereo-chemical parameters were calculated by PROCHECK (21) and WHAT_CHECK 194 (18) and fell within the range expected for a structure with similar resolution. The 195 crystallographic and model statistics for the di-zinc form of the wild-type and N220G mutant 196 structures are shown in Table 2. 197 198 Di-zinc form of the wild-type CphA metallo- -lactamase 199 The crystal structure of the di-zinc form of the wild-type CphA enzyme was solved by 200 molecular replacement using the structure of the mono-zinc form (PDB code 1X8G) as the 201 starting model. The structure was refined to a resolution of 2.03 Å. The Rwork and Rfree values for 202 the refined structure were 0.1589 and 0.2020, respectively. The crystals adopted a C2221 space 203 group with one molecule in the asymmetric unit. Only one residue (Ala195) was found in a 204 disallowed region of the Ramachandran plot. Ala195 (= 105°, = 155°) is located on the loop 205 between strands 8 and 9, with His196 at its apex. 206 The model of the wild-type di-zinc structure includes 226 amino acid residues (41-312, 207 according to the BBL numbering (13)). The last serine residue at the C-terminus is missing. The 208 electron density was good enough to identify three zinc ions (with two in the active site and one 209 on the surface) and a sulphate ion in the active site. The composition of the visible solvent sphere 210 is mentioned in table 2. The Gly232–Phe236 mobile loop region had a low electron density and 211 the main chain for the residues was modelled in two conformations. The Phe236 side chain was 10 212 not visible in the electron density map and other side chains were modelled with alternative 213 conformations. 214 CphA shows the typical fold of the metallo--lactamase superfamily. The overall 215 fold is not altered by the presence of the second zinc. The di-zinc form has an average RMSD 216 value of 0.272 Å for backbone atoms with respect to the mono-zinc form. 217 The positions of the zinc ions were verified at the level of the electron density and by using 218 anomalous signals. Both zinc ions were refined to a 100 % occupancy and show an increased 219 mobility. This is indicated by the B-factors (17.68 and 20.63 for the first and the second zinc 220 ions, respectively) lying over the mean B-factor (9.81). The first zinc ion is found in the Asp120- 221 Cys221-His263 site or “cysteine” site as previously observed in the mono-zinc form (14) (Figure 222 1). The distances between the zinc ion and the Asp120 carboxyl oxygen atom, the Cys221 223 sulphur atom, and the His263 side-chain nitrogen atom are 2.00, 2.26 and 2.09 Å, respectively. In 224 the mono-zinc form, these distances were 1.96, 2.20 and 2.05 Å, respectively (14). The 225 tetrahedral coordination sphere is completed by a sulphate ion from the crystallization solution at 226 a distance of 2.04 Å. 227 The second zinc ion is coordinated by the His118 and His196 residues. Both histidine 228 residues belong to the conserved “histidine” site in metallo--lactamases. The distances between 229 the zinc ion and the His118 ND1 and the His196 NE2 are 2.01 and 2.03 Å, respectively. In 230 CphA, the third ligand His116 is not conserved and is replaced by an asparagine residue, which is 231 not involved in the binding of the second zinc (the distance is more than 4 Å). A sulphate ion 232 (1.95 Å) and a water molecule (2.01Å) complete the vacant coordination positions of the zinc ion 233 in the “histidine” site, forming a tetrahedral coordination sphere. The sulphate ion acts as a 234 bridging agent between the zinc ions (Figure 1). The distances between the sulphate ion and zinc 11 235 in the “cysteine” site and zinc in the “histidine” site are 2.04 and 1.95 Å, respectively. The 236 distance between both zinc ions is 4.08 Å. 237 The third observed zinc ion was found on the surface, away from the active site (about 17 238 Å). It is coordinated by His289 (2.09 Å from His NE) and the coordination sphere is completed 239 by three chloride ions (2.19, 2.25 and 3.17 Å). 240 241 Di-zinc form of the N220G mutant 242 The crystal structure of the di-zinc form of N220G was solved by molecular replacement 243 based on the mono-zinc structure (PDB code 1X8G). The structure was refined to 1.70 Å with 244 Rwork and Rfree values of 0.1308 of 0.1602, respectively. 245 The model of the N220G di-zinc structure included all the 227 residues of the protein (41- 246 313 according to the BBL numbering (13)), three zinc ions (two in the active site and one on the 247 surface) and one sulphate ion located in the active site. The complete content of the solvent 248 sphere is described in table 2. The C-terminal residues were highly flexible. Ala195 was found in 249 a disallowed region of the Ramachandran plot. The electron density for the Gly232–Phe236 250 mobile loop region allowed a well-defined model to be constructed. 251 The active site of the N220G mutant with two bonded zinc ions had a conformation nearly 252 identical to that of the wild-type di-zinc enzyme. The RMSD for all main chain atoms was 0.216 253 Å. In contrast to the mono-zinc form of the N220G mutant where the zinc ion occupied two sites 254 1.5 Å apart (14), in the di-zinc form of the enzyme described here, the zinc ion was in the 255 classical “cysteine” site with distances to ligands identical to those in the wild-type structure. 256 Also, a sulphate ion and a water molecule (both with 50 % occupancy) were included in the 257 coordination sphere at distances of 2.04 and 2.27 Å, respectively. Under the new crystallisation 12 258 conditions described above, a mono-zinc form of the N220G mutant was also obtained, in which 259 a fully occupied zinc ion was present in the canonical “cysteine” site (data not shown). 260 The second zinc ion could not be modelled with an occupancy higher than 0.8. This 261 second zinc ion shows a B-factor of 9.85, nearly the same as the mean B-factor (7.84) and in the 262 same range as the B-factor of the zinc ion in the “cysteine” site (10.50). This zinc ion was 263 coordinated by His118 and His196. The coordination sphere was completed by a water molecule 264 (2.02 Å) and the sulphate ion (2.15 Å). An additional water molecule was visible at a distance of 265 2.54 Å. 266 The third zinc ion was found nearly in the same position as in the wild-type structure. 267 Here, the zinc ion and the coordinating chloride ions exhibited 50 % occupancy. The coordination 268 sphere was affected by a water molecule and a disordered side chain from Glu292. 269 270 Comparison between the mono- and di-zinc structures of CphA 271 The binding of the second zinc ion in the active site has no influence on the global 272 structure of CphA. This is confirmed by the low RMSD values obtained by superposition of the 273 mono- and di-zinc forms. The differences between the mono- and di-zinc structures are limited to 274 the Gly232–Asn233 loop region (Figure 2). This loop, located at the entrance to the active site, is 275 known to be stabilized upon the binding of the substrate (14) or inhibitors (19, 22). For the wild- 276 type di-zinc structure, this loop can exist in two conformations (both half occupied), 277 corresponding, respectively, to the open form (as in the mono-zinc structures 1X8G and 1X8H) 278 or the closed form (as in the structures with substrate or inhibitor in the active site, 1X8I, 2QDS 279 and 2GKL). In the di-zinc form of the N220G mutant, this loop is found in the closed form 280 (Figure 2). The closed conformation is stabilized by additional H-bonds between the Asn233 side 281 chain and the Ser235 side chain oxygen and backbone nitrogen. 13 282 The active site of the di-zinc forms can be superimposed nearly perfectly over all the 283 previously available CphA structures (PDB codes 1X8G, 1X8H, 1X8I, 2QDS and 2GKL). For 284 example, the RMSD is 0.56 Å for the superposition of the “cysteine” and “histidine” sites over 285 their counterparts in 1X8G. The position of the zinc in the “cysteine” site is nearly identical, with 286 the exception of the mono-zinc N220G mutant where a disordered zinc ion was observed (14). 287 The coordinating His118 residue in the “histidine” site is not affected by the binding of the 288 second zinc. In contrast, the His196 residue displays a slight flexibility. It is noticeable that the 289 orientation of the imidazole ring of His196 in all X-ray structures is selected to achieve an 290 optimal pattern of contacts in the coordination sphere (H-bonds, zinc binding). Therefore, in di- 291 zinc structures, the His196 imidazole ring is rotated by 180° compared to its position in the 292 mono-zinc structures (Figure 2). 293 294 Comparison with the active sites in B1 and B3 metallo--lactamases 295 The active sites of all three metallo-β-lactamase subclasses show high degree of 296 correlation (RMSD values between 0.5 Å and 1.6 Å). In subclass B2, the coordination sphere of 297 the zinc in the “histidine” site is altered by the substitution of His116 by an asparagine residue. In 298 subclasses B1 and B3, the His116 residue is involved in the binding of the zinc ion in the 299 “histidine” site (Figure 3). Removing one coordinating residue causes this zinc ion to shift by 300 1.12, 1.03, 1.03 and 0.92 Å compared to BcII (PDB code 1BVT) (Figure 3), VIM-2 (PDB code 301 1KO3), VIM-2 (PDB code 1KO2) and L1 (PDB code 2FM6) structures, respectively. The 302 position of the zinc in the “cysteine” site is nearly unchanged when compared to the available 303 structures of subclass B1 enzymes. 304 305 Stability study 14 306 As already reported (16), the helical contents of the apo-, mono- and di-zinc forms of the 307 wild-type CphA enzyme are similar. The apparent Tm values determined by CD spectroscopy 308 were 46.9, 58.7 and 61.6°C for the apo-, mono- and di-zinc forms, respectively. The mono-zinc 309 wild-type CphA is also less stable than the di-zinc form with urea as a denaturing reagent. 310 Transitions between native and denatured states occurred at 3.4 and 4.4 M urea for the mono- and 311 di-zinc forms, respectively. (data not shown) 312 The far-UV CD spectra of the apo-, mono- and di-zinc forms of the N220G mutant 313 exhibited small but significant differences (Figure 4a). However, as observed for the wild-type 314 enzyme, the helical content did not vary significantly. Thermal denaturation was irreversible in 315 all cases. The apparent Tm values determined by CD spectroscopy were 49.2, 61.7 and 62.9°C for 316 the apo-, mono- and di-zinc forms, respectively (Figure 4b). Also, the apparent Tm value 317 determined for the mono-zinc form of N220G using a thermal shift assay (Figure 4c) and by DSC 318 was 62°C but decreased to 50°C in the presence of 10 mM EDTA (Figure 4c). The mono-zinc 319 form of the N220G mutant was slightly less stable than the di-zinc form with urea as the 320 denaturing reagent. Transitions between native and denatured states occurred at 4.1 and 4.7 M 321 urea for the mono- and di-zinc forms, respectively (data not shown). 322 323 Is the “closed” position of the Gly232-Asn233 loop important for the inhibition by the second 324 zinc ion? - Analysis of the N233A mutant 325 An Asn233 residue is present in all metallo--lactamases with the exception of the L1, 326 BlaB and IND-1 enzymes (13). In CphA, the binding of a carbapenem substrate or inhibitor 327 modifies the Asn233 angle so that its side chain closes the entrance of the active site in a 328 conformation stabilised by the formation of an H-bond between the oxygen of the Asn233 side- 15 329 chain and the hydroxyl of Ser235 (14, 19, 22). In the structure of the di-zinc form of CphA, the 330 loop adopts the closed position even in the absence of substrate, thus hindering access to the 331 active site (Figure 2). To explore the role of the Asn233 residue in the inhibition phenomenon, 332 the N233A mutant was expressed in Escherichia coli BL21 (DE3) pLysS Star, purified to 333 homogeneity and characterized. The mass of the protein was verified by electrospray ionisation 334 mass spectrometry (ESI-MS). Within experimental error, the mutant was found to exhibit the 335 expected mass (25144 Da measured vs. 25146 Da expected). The far UV CD spectrum of the 336 mutant enzyme showed the same / ratio as the wild type. The enzyme was stored in 15mM 337 sodium cacodylate (pH 6.5) with < 0.4 µM free zinc. Under these conditions, the N233A protein 338 contained one zinc ion per molecule as reported for the wild-type -lactamase. The activity of the 339 mutant was measured in the absence of added Zn2+ (< 0.4 µM) with imipenem, nitrocefin and 340 CENTA (Table 3). The N233A mutant showed an increased Km value for imipenem. In contrast 341 to the wild-type enzyme, the N233A mutant was able to hydrolyse (albeit rather poorly) both 342 cephalosporins, with quite low Km values. The hydrolysis of imipenem by N233A was inhibited 343 by the binding of the second zinc ion, but the KD2 value decreased to 11 ± 2 µM compared to 46 344 µM for the wild-type enzyme. 345 16 346 Discussion 347 There are three subclasses of metallo--lactamases (B1, B2 and B3), all of which require 348 Zn2+ for activity and can bind either one or two zinc ions (12, 13). Subclass B2 metallo-- 349 lactamases are active only in the mono-zinc form, because the binding of a second zinc ion 350 inhibits the enzyme in a non-competitive manner (16). This behaviour contrasts with that of B1 351 and B3 subclasses, where the presence of a second zinc ion in the active site has either little effect 352 or facilitates enzyme activity. Since it has thus far proven difficult to determine the structure of 353 the di-zinc form of a subclass B2 -lactamase to investigate the basis of this inhibitory effect, we 354 set out to crystallise the di-zinc form of Aeromonas hydrophila CphA and solve its structure. 355 Previous attempts to determine the crystal structure of di-zinc A. hydrophila CphA have 356 not been successful, even with a high concentration of zinc in the mother liquor (14). It was 357 proposed that this might reflect the conditions used to grow crystals and/or the presence of a 358 carbonate ion in the active site. Also, it was suggested that the binding of the second zinc ion 359 required a change of active site conformation impossible to obtain in the crystal. Indeed, nuclear 360 magnetic resonance (NMR) measurements indicate major conformation changes upon binding of 361 the second zinc ion (C. Damblon, personal communication). However, CD spectra showed that 362 the secondary structures of the mono-zinc forms of the wild-type (16) and N220G CphA enzymes 363 are only slightly different from those of the di-zinc forms. By modifying the crystallization 364 conditions, we succeeded in obtaining CphA crystals with two zinc ions in the active site. 365 Moreover, the overall conformation of the enzyme was not affected by the presence of a 366 second zinc ion. The di-zinc form of CphA was obtained by soaking the crystal into a solution 367 containing a zinc concentration well above the KD2 value. In the crystal, major movements 368 leading to the significant unfolding of the protein as observed by NMR are impossible. 17 369 370 In the mono-zinc form, a carbonate ion was present in the active site (14). In our structures, a sulphate ion from the crystallization solution was bound to both zinc ions. 371 As previously postulated (4, 34), the second inhibitory zinc ion is located in a slightly 372 modified “histidine” binding site with the two conserved His118 and His196 residues as ligands. 373 The Met146 residue, proposed as one of the inhibitory zinc ion ligands together with His118 in 374 the ImiS metallo--lactamase (6), was 12.54 Å from this ion in CphA and the sulphur atom points 375 in the opposite direction. Also, the space between the His118 and Met146 residues is occupied by 376 the backbone of the Asn114-His118 loop, connecting strand 6 and helix 2, and thus does not 377 allow the accommodation of a zinc ion. The sequence of ImiS is 96% identical to that of CphA, 378 so it would be remarkable if these few substitutions produced such a radically different 379 mechanism for the binding of the inhibitory zinc ion. 380 This “atypical” coordination sphere with only two of the three conserved histidine 381 residues (figure 3) could probably explain the rather high value of the dissociation constant for 382 the “histidine” site in CphA (46 µM) compared to that in subclasses B1 and B3. The value of the 383 dissociation constant for the “histidine” site is 1.8 nM for BcII (a subclass B1 enzyme) and 384 subclass B3 enzymes have high affinity constants for both binding sites (KD < 6 nM) (35). 385 Vanhove et al. proposed an explanation for the inhibition of subclass B2 enzymes by the 386 second zinc ion. The side-chain of the Cys221 which is essential for binding the first zinc ion 387 would be displaced by the binding of the second zinc ion (34). However, a comparison between 388 mono- and di-zinc structures showed that this residue was not displaced. On the basis of the 389 results obtained here, we can confirm that the binding of the inhibitory zinc ion to the 390 catalytically important His118 and His196 residues prevents them from playing their catalytic 391 roles in the hydrolysis of carbapenems (4, 14) where His118 is the general base which activates 18 392 the hydrolytic water molecule and His196 contributes to the oxyanion hole by forming a H-bond 393 with the carbonyl oxygen of the -lactam bond (14). This model is supported by the very weak 394 activities of the H118A and H196A mutants (4). This conclusion reinforces the hypothesis that 395 the nucleophile is not adequately metal activated in B2 -lactamases (30, 36). Superimposition of 396 the structures of the dizinc forms of BcII (B1) and CphA shows that in the latter, the water 397 molecule does not bridge the two zinc ions but is only in interaction with that in the “histidine” 398 site (figure 5). It is possible that an interaction with this sole zinc ion does not sufficiently 399 decrease the water pKa so that the concentration of OH- ions remains very low. Moreover, 400 His118 which now serves as a zinc ligand can non longer play the role of general base. The 401 closure of the Gly232–Asn233 loop in the di-zinc form could also help to inhibit the enzyme, but 402 Asn233 probably plays only a minor role since the N233A mutant is still inhibited by the binding 403 of the second zinc ion. Finally, a third zinc ion is bound to a superficial histidine residue, but this 404 is unlikely to be involved in the inhibition phenomenon. Its binding is probably due to the high 405 zinc concentration utilized. 406 Whereas the N233S mutant of ImiS exhibits kinetic parameters similar to those of wild- 407 type enzyme (30), the N233A mutant of CphA shows an increased Km value for imipenem, and 408 thus seems to play a role in the binding of this substrate. Two-thirds of all sequenced metallo-β- 409 lactamases have an Asn at position 233 (12, 13), and this residue was shown to be involved in 410 substrate binding and activation by interacting electrostatically with the substrate -lactam 411 carbonyl (37). A same role for Asn233 residue in substrate binding by CphA was already 412 predicted by computational modelling (36) and is in agreement with our results. 413 In contrast to the zinc ion located in the “cysteine” site, the second zinc ion does not play 414 an important structural role. Indeed, the binding of the first zinc ion stabilizes the wild-type and 19 415 N220G CphA enzymes significantly, whereas the second zinc ion has a much more limited 416 impact on stability. Both the mono- and di-zinc forms of the N220G mutant are somewhat more 417 stable than their wild-type counterparts, perhaps explaining why crystals of the N220G mutant 418 are obtained more easily than those of the wild-type enzyme. 419 In conclusion, the catalytic metal ion is located in the “cysteine” site of CphA and the 420 second inhibitory zinc ion binds to a slightly modified “histidine” site. The “histidine” site has 421 first been considered as the catalytic site in the mononuclear metallo--lactamases. Indeed, in the 422 first reported monozinc structures (B1 enzymes such as BcII, VIM-2, VIM-4 and SPM-1), the 423 single metal ion was shown to be located in the “histidine” site (5, 15, 25). In contrast, a B3 424 enzyme called GOB was shown to be active as a monozinc enzyme with the metal ion located in 425 the “cysteine” site (24). Recently, Vila and co-workers proposed that B1 enzymes can be active 426 in their mono- and dizinc forms and that in mono-cobalt BcII the metal ion is localized in the 427 “cysteine” site (23, 31). Two mononuclear mutants of BcII in which each of the metal binding 428 sites was selectively removed produced inactive variants. The authors concluded that the 429 mononuclear form can be active only if assisted by residues at the other binding site (1). 430 According to the model proposed by Tioni et al. (31), the metal ion in the “histidine” site would 431 activate the hydrolytic water molecule in the dizinc enzyme. Vila and co-workers proposed that 432 this is facilitated by a net of H-bond interactions in the mononuclear enzyme (23, 31) probably 433 including His118 or Asp120, as already proposed for CphA (4, 14, 36). However, Asp120 434 appears unlikely because it already coordinates a zinc ion. In subclass B2, as previously 435 postulated by Vanhove et al. (34), Asn116 is not involved in the binding of the second metal ion 436 but the N116H (34) and N116H-N220G (2) mutants could have a reconstituted His116-His118- 437 His196 site and behave similarly to B1 enzymes. Indeed, the N116H-N220G mutant has an 438 extended substrate spectrum and its di-zinc form is active (2). 20 439 The crystal structure of the subclass B2 CphA -lactamase in its metal-inhibited form 440 provides thus an answer to the structural characterization of the inhibitory site that has been 441 elusive and controversial so far. 442 21 443 Acknowledgements 444 The work in Liège was supported by the Belgian Federal Government (PAI P5/33) and 445 grants from the FNRS (Brussels, Belgium, FRFC grant n° 2.4511.06 and Lot. Nat. 9.4538.03). 446 C.B. is a FRS/FNRS post-doctoral researcher. 447 22 448 References 449 1. Abriata, L. A., L. 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Composition of the two metal-binding sites in the three metallo--lactamase 557 subclasses. * This “histidine” site is still putative, since the 3D structure of the GOB enzyme is 558 not available. 559 Enzymes "Histidine" site "Cysteine" site B1 MLs His116-His118-His196 Asp120-Cys221-His263 B2 MLs His118-His196 Asp120-Cys221-His263 B3 MLs His116-His118-His196 Asp120-His121-His263 B3 ML GOB (Gln116)-His118-His196* Asp120-His121-His263 560 28 561 Table 2. X-ray data collection and structure refinement 562 CphA WT di-zinc CphA N220G di-zinc A. Data Collection statistics Resolution range (Å) Wavelength (Å) Unit cell parameters Space group a (Å) b (Å) c (Å) Rsym No. observed reflections No. unique reflections Completeness (%) I/sigma(I) Multiplicity B (Wilson) B. Refinement statistics No. reflections used Molecules/asymmetric unit Rworking Rfree Residues in disallowed region RMS deviation from ideal Bonds (Å) Angles (deg.) Mean B factor No. atoms (non-H) No. atoms (protein) No. of H20 molecules Other molecules/ions Zn2+ SO4 CO3 ClGlycerol 19.7-2.03 1.5418 19.6-1.70 1.5418 C2221 42.80 101.05 116.49 0.062 235065 16754 99.7 34.4 14 15.69 C2221 42.68 101.06 116.83 0.032 206898 28046 99.7 42.1 7.4 11.96 16754 1 0.1568 0.2020 1 28046 1 0.1388 0.16024 1 0.011 1.217 9.81 2090 1850 202 0.011 1.550 7.84 2198 1882 252 3 4 2 7 - 3 7 7 3 563 29 564 Table 3. Kinetic parameters of the wild-type and N233A CphA enzymes. All the 565 experiments were performed at 30°C in 15 mM cacodylate (pH 6.5), [Zn2+] < 0.4 µM. Standard 566 errors were below 10%. NH: no observed hydrolysis 567 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 14 250 568 30 569 Figure legends 570 571 Figure 1 572 Structure of the wild-type CphA metal-binding sites. Zinc ions and water molecule are shown 573 as grey and red spheres, respectively. ZnHis is the zinc ion that binds in the “histidine” site while 574 ZnCys is the zinc ion that binds in the “cysteine” site. The anomalous map at a contour level of 4 575 is shown in orange. Relevant distances between zinc ions and ligands are indicated. 576 577 Figure 2 578 Superposition of the mono- (in grey) and di-zinc (in yellow) forms of the N220G CphA 579 enzyme. The Gly232–Asn233 loop region of the di-zinc form is represented in red to underline 580 the conformational change. The positions of the His196 residue in the mono-form (H196mono) and 581 in the di-zinc form (H196di) are represented. The zinc ions in the “histidine” site (ZnHis) and in the 582 “cysteine” site (ZnCys) are shown as grey spheres. The distance between His196di and the zinc ion 583 located in the “histidine” site is indicated. 584 585 Figure 3 586 The “histidine” site in CphA (a) and in BcII (PDB code 1BVT) (b). Zinc ions and water 587 molecules are represented as grey and red spheres, respectively. 588 589 Figure 4 590 Structural studies of the N220G mutant 31 591 592 a) Circular dichroism spectra of the apo-enzyme (dotted line), mono-zinc (dashed line) and di-zinc forms (solid line) of the N220G enzyme (0.3 mg/ml in both cases). 593 b) Thermal denaturation of the N220G mutant (0.5 mg/ml): fraction of native protein vs 594 temperature (°C). Apo-N220G (dotted line), mono-zinc N220G (dashed line), di-zinc 595 N220G (solid line). 596 597 c) Thermal shift assay for N220G (0.3 mg/ml). N220G + 10 mM EDTA (dotted line), monozinc N220G (dashed line). 598 599 Figure 5 600 Superimposition of the structures of the di-zinc forms of BcII (PDB code 2BFK) and CphA. 601 Zinc ligands of BcII and CphA are represented as grey and green sticks, respectively. Zinc ions of 602 BcII and CphA are shown as grey and green spheres, respectively. ZnHis is the zinc ion that binds 603 in the “histidine” site while ZnCys is the zinc ion that binds in the “cysteine” site. Water molecules 604 of BcII and CphA are shown as purple and red spheres, respectively. The sulphate ion that 605 bridges the two zinc ions in the CphA structure (SO4CphA) is also represented, as well as glycerol, 606 the fifth ligand of ZnCys in BcII (glycerolBcII). 607 608 32