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The structure of the di-zinc subclass B2 metallo--lactamase CphA reveals
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that the second inhibitory zinc ion binds in the “histidine” site.
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Carine Bebrone1,3,*,§, Heinrich Delbrück2,*, Michaël B. Kupper2, Philipp Schlömer2,
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Charlotte Willmann2, Jean-Marie Frère1, Rainer Fischer2, Moreno Galleni3 and Kurt M. V.
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Hoffmann2
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Liège, Belgium
Centre for Protein Engineering, University of Liège, Allée du 6 Août B6, Sart-Tilman 4000
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Forckenbeckstrasse 6, 52074 Aachen, Germany
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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,
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both authors contributed equally to this work
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Running title: The inhibitory zinc ion binds in the “histidine” site of CphA
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§
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Allée du 6 Août B6, Sart-Tilman, 4000 Liège, Belgium, Tel.: 003243663315, Fax:
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003243663364, e-mail: [email protected]
Corresponding author: Carine Bebrone, Centre for Protein Engineering, University of Liège,
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Abstract
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Bacteria can defend themselves against -lactam antibiotics through the expression of
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class B -lactamases, which cleave the -lactam amide bond and render the molecule harmless.
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There are three subclasses of class B -lactamases (B1, B2 and B3), all of which require Zn2+ for
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activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo--lactamases
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are most active as di-zinc enzymes, subclass B2 enzymes such as Aeromonas hydrophila CphA
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are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to
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determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals
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allowed us to solve the crystal structures of the di-zinc forms of the wild-type enzyme and
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N220G mutant. The first zinc ion binds in the “cysteine” site, as previously determined for the
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mono-zinc form of the enzyme. The second zinc ion occupies a slightly modified “histidine” site,
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where the conserved His118 and His196 residues act as metal ligands. This atypical coordination
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sphere probably explains the rather high dissociation constant for the second zinc ion compared
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to those observed in enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results
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from immobilization of the catalytically-important His118 and His196 residues, as well as the
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folding of the Gly232–Asn233 loop into a position that covers the active site.
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Introduction
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Class B -lactamases (also called zinc -lactamases or metallo--lactamases) play a key
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role in bacterial resistance to -lactam antibiotics by efficiently catalyzing the hydrolysis of the
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-lactam amide bond. The existence of such enzymes is a particular concern because they are
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effective against most -lactam antibiotics (including the carbapenems), the corresponding genes
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are easily transferred between bacteria and there are no clinically useful inhibitors. On the basis
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of the known sequences, three different lineages, identified as subclasses B1, B2, and B3, can be
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characterized (12, 13). All class B -lactamases possess two potential zinc-binding sites and
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share a small number of conserved motifs bearing some of the residues that coordinate the zinc
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ion(s), notably His/Asn/Gln116-Xaa-His118-Xaa-Asp120 and Gly/Ala195-His196-Ser/Thr197
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(13). Structural analysis of subclass B1 enzymes shows that one zinc ion has a tetrahedral
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coordination sphere involving His116, His118, His196 and a water molecule or OH- ion
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(“histidine” site), whereas the other has a trigonal-pyramidal coordination sphere involving
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Asp120, Cys221, His263 and two water molecules (“cysteine” site) (Table 1). One
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water/hydroxide serves as a ligand for both metal ions (3). In the mononuclear structures of B1
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enzymes (BcII, VIM-2, SPM-1 and VIM-4), the sole metal ion was found to be located in the
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“histidine” site (5, 15, 25 and P. Lassaux, unpublished data). In some monozinc B1 structures, the
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Cys221 residue is found under an oxidized form. In subclass B3, the “histidine” site is similar to
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that found in subclass B1, but Cys221 is replaced by a serine residue and the second zinc ion is
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coordinated by Asp120, His121, His263 and the nucleophilic water molecule which again forms
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a bridge between the two metal ions (Table 1) (33).
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Aeromonas hydrophila CphA is a subclass B2 metallo--lactamase characterized by a
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uniquely narrow specificity. CphA efficiently hydrolyses only carbapenems and shows very poor
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activity against penicillins and cephalosporins, in contrast to subclass B1 and B3 enzymes which
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hydrolyse nearly all -lactam compounds, with the exception of monobactams (10, 29). In
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contrast to subclass B1 and B3 enzymes which are more active as di-zinc species, subclass B2 is
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inhibited in a non-competitive manner by the binding of a second zinc ion. For CphA, the
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dissociation constant of the second zinc ion (KD2) is 46 µM at pH 6.5 (16). In agreement with
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EXAFS studies (17) and site-directed mutagenesis (34), the crystallographic structure of CphA
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shows that the first zinc ion is in the “cysteine” site (14). However, the second binding site in
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subclass B2 enzymes remains to be determined because Garau and colleagues could not produce
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crystals of the di-zinc form despite presence of zinc concentration well above KD2 (14). The
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structure of Sfh-1, a subclass B2 enzyme from Serratia fonticola, has been solved recently, once
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again with only one zinc ion in the active site (11). In subclass B2, the His116 residue found
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throughout the metallo--lactamase superfamily (with the exception of the B3 GOB enzymes
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where Gln is present) is replaced by an asparagine (8, 12, 13). It has been previously shown that
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the Asn116 residue has no role in the binding of the zinc ions in CphA (34). On the basis of
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spectroscopic studies with the Aeromonas veronii cobalt-substituted ImiS enzyme, Crawford and
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co-workers postulated that the second metal binding site was not the traditional “histidine” site
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but a site, remote from the active site, which involves both His118 and Met146 as zinc ligands (6,
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7). However, a recent study of potential zinc ligand mutants contradicts this hypothesis and
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indicates that the position of the second zinc ion in CphA is probably equivalent to the “histidine”
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site observed in subclasses B1 and B3 enzymes, with His118 and His 196 involved in the binding
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of this second zinc perhaps with Cys221 (or Asn116 or Asp120) as the third ligand (4).
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Since crystals of the wild-type CphA protein could not be obtained directly, single-site
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mutants were engineered by site-directed mutagenesis and overproduced. These mutants were
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selected in order to introduce residues that are conserved in either subclass B1 or B3, or both (2).
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Among these mutants, the N220G mutant (2) easily yielded crystals which were used as seeds to
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grow wild-type CphA crystals (14). The kinetic parameters of the wild-type and N220G mutant
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CphA enzymes were similar, indicating strong conservation of enzymatic properties in the mutant
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(2). However, the N220G mutation results in a slightly higher KD2 value (86 µM) (2). We
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therefore set out to solve the crystallographic structures of the di-zinc forms of the wild-type
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CphA enzyme and its N220G mutant. We confirmed that the second zinc ion occupies a slightly
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modified “histidine” site, where the conserved His118 and His196 residues act as metal ligands.
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The implication of this discovery on what is known about the coordination of zinc ions by
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metallo--lactamases is discussed. We propose an explanation for the inhibition by the second
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zinc ion. Moreover, the role of the Asn233 residue in this inhibition phenomenon is also
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apprehended based on a site-directed mutagenesis study. In this work, a structural role for the
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zinc ions is also investigated.
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Materials and Methods
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Protein purification and crystallization
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Site-directed mutagenesis, protein expression and purification of the proteins were carried
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out as described previously (2), with the addition of a third purification step consisting of a size
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exclusion chromatography (Hiload 16/60 Superdex 75 prep grade column, Pharmacia Biotech).
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The purified enzyme solution was dialyzed against 15 mM sodium cacodylate (pH 6.5).
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Monodispersity of the protein solutions and the hydrodynamic radii of the dissolved protein
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molecules were checked by Dynamic Light Scattering (DLS). The crystallisation conditions used
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previously (14) were not easy to reproduce because the crystallisation drop comprised two
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phases. Therefore, a screen was carried out to find new conditions. Initial screens were carried
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out at 8°C using commercial Hampton Crystal Screens 1 and 2, Grid Screen Ammonium
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Sulphate and Grid Screen PEG 6000 (Hampton Research, California, USA). N220G CphA (10
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mg/ml) was crystallized from 2.2–2.4 M ammonium sulphate and 0.1 M MES (pH 6.0–6.5),
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using the sitting drop method with new crystallisation plates designed by Taorad GmbH (Aachen,
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Germany). The (1 µl) reservoir solution was mixed with the protein solution (1 µl) and the
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mixture was left to equilibrate against the solution reservoir. Typically, orthorhombic crystals of
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the N220G mutant grew within a few days to dimensions of 80 x 100 x 100 µm and belong to
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space group C2221 (a=42.68, b=101.06 and c=116.82). Crystals of the wild-type CphA enzyme
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were obtained under similar conditions using mutant micro-crystals as starting seeds and showed
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similar orthorhombic symmetry in space group C2221 (a=42.80, b=101.50 and c=116.49). Before
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data collection, 1µl of 100 mM ZnCl2 was added to the drops containing the wild-type and the
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mutant crystals and soaked for one day.
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Data collection and processing
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For data collection, wild-type and mutant crystals were transferred to a cryoprotectant
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solution containing reservoir solution supplemented with 30% (v/v) glycerol. The mounted
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crystals were flash-frozen in a liquid nitrogen stream. Near-complete X-ray data sets were
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collected using a Bruker FR591 rotating anode X-ray generator and a Mar345dtb detector.
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Diffraction data were processed with XDS (20) and scaled with SCALA from the CCP4-Suite
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(32).
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Structure determination and refinement
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The structures of di-zinc variants from the wild-type and N220G mutant enzymes were
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solved using a molecular replacement approach with Molrep (32), and the mono-zinc structure of
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CphA (PDB file 1X8G) as starting model. The resulting model was rebuilt with ARP/wARP (28).
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The structures were refined in a cyclic process including manual inspection of the electron
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density with Coot (9) and refinement with Refmac (26). The refinement to convergence was
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carried out with isotropic B-values and using TLS parameter (27). The positions of the zinc,
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sulphate and chloride ions were verified by calculating anomalous maps and the occupancies of
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the ions were determined by disappearing of difference electron density. Alternative
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conformations were modelled for a number of side chains and occupancies were adjusted to yield
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similar B-values for the disordered conformations.
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Circular dichroism (CD) spectroscopy
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Circular dichroism (CD) spectra for the apo-, mono-and di-zinc forms of the wild-type
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and N220G enzymes (0.3 mg/ml) were obtained using a JASCO J-810 spectropolarimeter. The
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apoenzymes were obtained in the presence of 10 mM EDTA. The di-zinc forms were obtained in
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the presence of 500 µM Zn2+. The spectra were scanned at 25°C with 1 nm steps from 200 to 250
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nm (far UV). Thermal stability was assessed for the apo-, mono- and di-zinc forms of the wild-
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type and N220G proteins using CD spectroscopy at 220 nm with increasing temperature (25°C–
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85°C). Urea denaturation studies of the wild-type and N220G enzymes were also carried out in
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the presence and absence of 500 µM Zn2+. The mono- and di-zinc enzymes were incubated for
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18h in increasing concentrations of urea (0–8 M) at 4°C. Stability was assessed for the mono- and
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di-zinc forms of both proteins using CD spectroscopy at 220 nm with increasing concentrations
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of urea.
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Thermal shift assay
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Thermal shift assays were carried out using a LightCycler real-time PCR instruments
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(Roche Diagnostics). Briefly, 10 µl of wild-type CphA enzyme or the N220G mutant (0.3 mg/ml)
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was mixed with 10 µl of 5000 x Sypro Orange (Molecular Probes) diluted 1:500 in 15 mM
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cacodylate (pH 6.5). Samples were heat-denatured from 25 to 90°C at a rate of 0.5°C per minute.
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Protein thermal unfolding curves were monitored by detecting changes in Sypro Orange
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fluorescence. The inflection point of the fluorescence vs temperature curves was identified by
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plotting the first derivative over the temperature and the minima were referred to as the melting
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temperature (Tm). Buffer fluorescence was used as the control.
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Differential Scanning Calorimetry (DSC)
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Thermal denaturation was investigated using a NDSC 6100 differential scanning
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calorimeter (Calorimetry Sciences Corp., Lindon, USA) after dialysis of the N220G enzyme
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sample (1 mg/ml) against 15 mM cacodylate (pH 6.5). The dialysis buffer was used as reference.
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The data were collected and analysed with a MCS Observer software package assuming a two-
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state folding mechanism.
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Analysis of the N233A mutant
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The Quik Change Site-directed mutagenesis kit (Stratagene Inc., La Jolla, Calif.) was used
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to introduce the N233A mutation into CphA, using pET9a-CphA WT as the template, forward
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primer
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CGGCAAAGCTCAGGGCGCCCAGCTTCTCC-3′. The vector was then introduced into E. coli
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strain BL21 (DE3) pLysS Star. Overexpression and purification of the mutant protein, CD
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spectroscopy, electrospray ionization-mass spectrometry (ESI-MS) and the determination of
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metal content, kinetic parameters and residual activity in the presence of increasing
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concentrations of zinc were carried out as previously described (2, 4, 34).
5′-GGAGAAGCTGGGCGCCCTGAGCTTTGCCG-3′
and
reverse
primer
5′-
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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).
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Results
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Structure determination.
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The crystal structures of the zinc saturated wild-type and N220G CphA enzymes were
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solved. Stereo-chemical parameters were calculated by PROCHECK (21) and WHAT_CHECK
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(18) and fell within the range expected for a structure with similar resolution. The
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crystallographic and model statistics for the di-zinc form of the wild-type and N220G mutant
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structures are shown in Table 2.
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Di-zinc form of the wild-type CphA metallo- -lactamase
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The crystal structure of the di-zinc form of the wild-type CphA enzyme was solved by
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molecular replacement using the structure of the mono-zinc form (PDB code 1X8G) as the
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starting model. The structure was refined to a resolution of 2.03 Å. The Rwork and Rfree values for
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the refined structure were 0.1589 and 0.2020, respectively. The crystals adopted a C2221 space
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group with one molecule in the asymmetric unit. Only one residue (Ala195) was found in a
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disallowed region of the Ramachandran plot. Ala195 (= 105°, = 155°) is located on the loop
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between strands 8 and 9, with His196 at its apex.
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The model of the wild-type di-zinc structure includes 226 amino acid residues (41-312,
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according to the BBL numbering (13)). The last serine residue at the C-terminus is missing. The
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electron density was good enough to identify three zinc ions (with two in the active site and one
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on the surface) and a sulphate ion in the active site. The composition of the visible solvent sphere
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is mentioned in table 2. The Gly232–Phe236 mobile loop region had a low electron density and
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the main chain for the residues was modelled in two conformations. The Phe236 side chain was
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not visible in the electron density map and other side chains were modelled with alternative
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conformations.
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CphA shows the typical  fold of the metallo--lactamase superfamily. The overall
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fold is not altered by the presence of the second zinc. The di-zinc form has an average RMSD
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value of 0.272 Å for backbone atoms with respect to the mono-zinc form.
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The positions of the zinc ions were verified at the level of the electron density and by using
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anomalous signals. Both zinc ions were refined to a 100 % occupancy and show an increased
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mobility. This is indicated by the B-factors (17.68 and 20.63 for the first and the second zinc
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ions, respectively) lying over the mean B-factor (9.81). The first zinc ion is found in the Asp120-
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Cys221-His263 site or “cysteine” site as previously observed in the mono-zinc form (14) (Figure
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1). The distances between the zinc ion and the Asp120 carboxyl oxygen atom, the Cys221
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sulphur atom, and the His263 side-chain nitrogen atom are 2.00, 2.26 and 2.09 Å, respectively. In
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the mono-zinc form, these distances were 1.96, 2.20 and 2.05 Å, respectively (14). The
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tetrahedral coordination sphere is completed by a sulphate ion from the crystallization solution at
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a distance of 2.04 Å.
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The second zinc ion is coordinated by the His118 and His196 residues. Both histidine
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residues belong to the conserved “histidine” site in metallo--lactamases. The distances between
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the zinc ion and the His118 ND1 and the His196 NE2 are 2.01 and 2.03 Å, respectively. In
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CphA, the third ligand His116 is not conserved and is replaced by an asparagine residue, which is
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not involved in the binding of the second zinc (the distance is more than 4 Å). A sulphate ion
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(1.95 Å) and a water molecule (2.01Å) complete the vacant coordination positions of the zinc ion
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in the “histidine” site, forming a tetrahedral coordination sphere. The sulphate ion acts as a
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bridging agent between the zinc ions (Figure 1). The distances between the sulphate ion and zinc
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in the “cysteine” site and zinc in the “histidine” site are 2.04 and 1.95 Å, respectively. The
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distance between both zinc ions is 4.08 Å.
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The third observed zinc ion was found on the surface, away from the active site (about 17
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Å). It is coordinated by His289 (2.09 Å from His NE) and the coordination sphere is completed
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by three chloride ions (2.19, 2.25 and 3.17 Å).
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Di-zinc form of the N220G mutant
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The crystal structure of the di-zinc form of N220G was solved by molecular replacement
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based on the mono-zinc structure (PDB code 1X8G). The structure was refined to 1.70 Å with
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Rwork and Rfree values of 0.1308 of 0.1602, respectively.
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The model of the N220G di-zinc structure included all the 227 residues of the protein (41-
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313 according to the BBL numbering (13)), three zinc ions (two in the active site and one on the
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surface) and one sulphate ion located in the active site. The complete content of the solvent
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sphere is described in table 2. The C-terminal residues were highly flexible. Ala195 was found in
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a disallowed region of the Ramachandran plot. The electron density for the Gly232–Phe236
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mobile loop region allowed a well-defined model to be constructed.
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The active site of the N220G mutant with two bonded zinc ions had a conformation nearly
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identical to that of the wild-type di-zinc enzyme. The RMSD for all main chain atoms was 0.216
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Å. In contrast to the mono-zinc form of the N220G mutant where the zinc ion occupied two sites
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1.5 Å apart (14), in the di-zinc form of the enzyme described here, the zinc ion was in the
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classical “cysteine” site with distances to ligands identical to those in the wild-type structure.
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Also, a sulphate ion and a water molecule (both with 50 % occupancy) were included in the
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coordination sphere at distances of 2.04 and 2.27 Å, respectively. Under the new crystallisation
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conditions described above, a mono-zinc form of the N220G mutant was also obtained, in which
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a fully occupied zinc ion was present in the canonical “cysteine” site (data not shown).
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The second zinc ion could not be modelled with an occupancy higher than 0.8. This
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second zinc ion shows a B-factor of 9.85, nearly the same as the mean B-factor (7.84) and in the
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same range as the B-factor of the zinc ion in the “cysteine” site (10.50). This zinc ion was
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coordinated by His118 and His196. The coordination sphere was completed by a water molecule
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(2.02 Å) and the sulphate ion (2.15 Å). An additional water molecule was visible at a distance of
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2.54 Å.
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The third zinc ion was found nearly in the same position as in the wild-type structure.
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Here, the zinc ion and the coordinating chloride ions exhibited 50 % occupancy. The coordination
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sphere was affected by a water molecule and a disordered side chain from Glu292.
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Comparison between the mono- and di-zinc structures of CphA
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The binding of the second zinc ion in the active site has no influence on the global
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structure of CphA. This is confirmed by the low RMSD values obtained by superposition of the
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mono- and di-zinc forms. The differences between the mono- and di-zinc structures are limited to
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the Gly232–Asn233 loop region (Figure 2). This loop, located at the entrance to the active site, is
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known to be stabilized upon the binding of the substrate (14) or inhibitors (19, 22). For the wild-
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type di-zinc structure, this loop can exist in two conformations (both half occupied),
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corresponding, respectively, to the open form (as in the mono-zinc structures 1X8G and 1X8H)
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or the closed form (as in the structures with substrate or inhibitor in the active site, 1X8I, 2QDS
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and 2GKL). In the di-zinc form of the N220G mutant, this loop is found in the closed form
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(Figure 2). The closed conformation is stabilized by additional H-bonds between the Asn233 side
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chain and the Ser235 side chain oxygen and backbone nitrogen.
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The active site of the di-zinc forms can be superimposed nearly perfectly over all the
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previously available CphA structures (PDB codes 1X8G, 1X8H, 1X8I, 2QDS and 2GKL). For
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example, the RMSD is 0.56 Å for the superposition of the “cysteine” and “histidine” sites over
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their counterparts in 1X8G. The position of the zinc in the “cysteine” site is nearly identical, with
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the exception of the mono-zinc N220G mutant where a disordered zinc ion was observed (14).
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The coordinating His118 residue in the “histidine” site is not affected by the binding of the
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second zinc. In contrast, the His196 residue displays a slight flexibility. It is noticeable that the
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orientation of the imidazole ring of His196 in all X-ray structures is selected to achieve an
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optimal pattern of contacts in the coordination sphere (H-bonds, zinc binding). Therefore, in di-
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zinc structures, the His196 imidazole ring is rotated by 180° compared to its position in the
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mono-zinc structures (Figure 2).
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Comparison with the active sites in B1 and B3 metallo--lactamases
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The active sites of all three metallo-β-lactamase subclasses show high degree of
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correlation (RMSD values between 0.5 Å and 1.6 Å). In subclass B2, the coordination sphere of
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the zinc in the “histidine” site is altered by the substitution of His116 by an asparagine residue. In
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subclasses B1 and B3, the His116 residue is involved in the binding of the zinc ion in the
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“histidine” site (Figure 3). Removing one coordinating residue causes this zinc ion to shift by
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1.12, 1.03, 1.03 and 0.92 Å compared to BcII (PDB code 1BVT) (Figure 3), VIM-2 (PDB code
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1KO3), VIM-2 (PDB code 1KO2) and L1 (PDB code 2FM6) structures, respectively. The
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position of the zinc in the “cysteine” site is nearly unchanged when compared to the available
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structures of subclass B1 enzymes.
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Stability study
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As already reported (16), the helical contents of the apo-, mono- and di-zinc forms of the
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wild-type CphA enzyme are similar. The apparent Tm values determined by CD spectroscopy
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were 46.9, 58.7 and 61.6°C for the apo-, mono- and di-zinc forms, respectively. The mono-zinc
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wild-type CphA is also less stable than the di-zinc form with urea as a denaturing reagent.
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Transitions between native and denatured states occurred at 3.4 and 4.4 M urea for the mono- and
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di-zinc forms, respectively. (data not shown)
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The far-UV CD spectra of the apo-, mono- and di-zinc forms of the N220G mutant
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exhibited small but significant differences (Figure 4a). However, as observed for the wild-type
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enzyme, the helical content did not vary significantly. Thermal denaturation was irreversible in
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all cases. The apparent Tm values determined by CD spectroscopy were 49.2, 61.7 and 62.9°C for
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the apo-, mono- and di-zinc forms, respectively (Figure 4b). Also, the apparent Tm value
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determined for the mono-zinc form of N220G using a thermal shift assay (Figure 4c) and by DSC
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was 62°C but decreased to 50°C in the presence of 10 mM EDTA (Figure 4c). The mono-zinc
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form of the N220G mutant was slightly less stable than the di-zinc form with urea as the
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denaturing reagent. Transitions between native and denatured states occurred at 4.1 and 4.7 M
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urea for the mono- and di-zinc forms, respectively (data not shown).
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Is the “closed” position of the Gly232-Asn233 loop important for the inhibition by the second
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zinc ion? - Analysis of the N233A mutant
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An Asn233 residue is present in all metallo--lactamases with the exception of the L1,
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BlaB and IND-1 enzymes (13). In CphA, the binding of a carbapenem substrate or inhibitor
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modifies the Asn233  angle so that its side chain closes the entrance of the active site in a
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conformation stabilised by the formation of an H-bond between the oxygen of the Asn233 side-
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chain and the hydroxyl of Ser235 (14, 19, 22). In the structure of the di-zinc form of CphA, the
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loop adopts the closed position even in the absence of substrate, thus hindering access to the
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active site (Figure 2). To explore the role of the Asn233 residue in the inhibition phenomenon,
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the N233A mutant was expressed in Escherichia coli BL21 (DE3) pLysS Star, purified to
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homogeneity and characterized. The mass of the protein was verified by electrospray ionisation
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mass spectrometry (ESI-MS). Within experimental error, the mutant was found to exhibit the
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expected mass (25144 Da measured vs. 25146 Da expected). The far UV CD spectrum of the
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mutant enzyme showed the same / ratio as the wild type. The enzyme was stored in 15mM
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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. J Gonzalez, L. I. Llarrull, P. E. Tomatis, W. K. Myers, A. L. Costello,
450
D. L Tierney, and A. J. Vila. 2008. Engineered mononuclear variants in Bacillus cereus
451
metallo--lactamase are inactive. Biochemistry, 47:8590–8599.
452
2. Bebrone, C., C. Anne, K. De Vriendt, B. Devresse, J. Van Beeumen, J.M. Frère, and M.
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Galleni. 2005. Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA
454
metallo--lactamase by site-directed mutagenesis, J. Biol. Chem.,17:180-188.
455
3. Bebrone, C. 2007. Metallo--lactamases (classification, activity, genetic organization,
456
structure, zinc coordination) and their Superfamily. Biochem. Pharma. 74:1686-1701.
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458
Beeumen, O. Dideberg, J.M. Frère, and M. Galleni. 2008. Mutational analysis of the zinc and
459
substrate binding sites in the CphA metallo--lactamase from Aeromonas hydrophila, Biochem.
460
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461
5. Carfi, A., S. Parès, E. Duée, M. Galleni, C. Duez, J.M. Frère, and O. Dideberg. 1995. The
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3-D structure of a zinc metallo--lactamase from Bacillus cereus reveals a new type of protein
463
fold. EMBO J. 14:4914-4921.
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6. Costello, A.L., N.P. Sharma, K.W. Yang, M.W. Crowder, and D.L. Tierney. 2006. X-ray
465
absorption spectroscopy of the zinc-binding sites in the class B2 metallo--lactamase ImiS from
466
Aeromonas veronii bv. sobria. Biochemistry. 45:13650-13658.
467
7. Crawford, P.A., K.W. Yang, N. Sharma, B. Bennett, and M.W. Crowder. 2005.
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Spectroscopic studies on cobalt(II)-substituted metallo--lactamase ImiS from Aeromonas
469
veronii bv. sobria. Biochemistry. 44:5168-5176.
23
470
8. Daiyasu, H., K. Osaka, Y. Ishino, and H. Toh. 2001. Expansion of the zinc metallo-
471
hydrolase family of the -lactamase fold. FEBS Lett. 503:1-6.
472
9. Emsley, P., and K.Cowtan. 2004. Coot: model-building tools for molecular graphics. Acta
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Crystallogr. D60:2126-2132.
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10. Felici, A., G. Amicosante, A. Oratore, R. Strom, P. Ledent, B. Joris, L. Fanuel, and J.M.
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Frère. 1993. An overview of the kinetic parameters of class B -lactamases. Biochem.J.
476
291:151-155.
477
11. Fonseca, F., A. Correia, and J. Spencer. 2008. Structural and kinetic characterisation of
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Sfh-1, the B2 metallo--lactamase of Serratia fonticola. p.46. Proceedings of the 10th -
479
lactamase meeting, Eretria, Greece.
480
12. Galleni, M., J. Lamotte-Brasseur, G.M. Rossolini, J. Spencer, O. Dideberg, J.M. Frère,
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and the Metallo--lactamases Working Group. 2001. Standard numbering scheme for class B
482
-lactamases. Antimicrob.Agents Chemother., 45:660-663.
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13. Garau, G., I. Garcia-Saez, C. Bebrone, C. Anne, P.S. Mercuri, M. Galleni, J.M. Frère,
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and O. Dideberg. 2004. Update of the standard numbering scheme for class B -lactamases.
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Antimicrob.Agents Chemother., 48:2347-2349.
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14. Garau, G., C. Bebrone, C. Anne, M. Galleni, J.M. Frère, and O. Dideberg. 2005. A
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Metallo--lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase
488
CphA and its Complex with Biapenem. J. Mol. Biol. 345:785-795.
489
15. Garcia-Saez, I., J.D. Docquier, G.M. Rossolini, and O. Dideberg. 2008. The three-
490
dimensional structure of VIM-2, a Zn--lactamase from Pseudomonas aeruginosa in its reduced
491
and oxidised form. J. Mol. Biol. 375:604-611.
24
492
16. Hernandez-Valladares, M., A. Felici., G. Weber, H.W. Adolph, M. Zeppezauer, G.M.
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Rossolini, G. Amicosante, J.M. Frère, and M. Galleni. 1997 Zn(II) dependence of the
494
Aeromonas hydrophila AE036 metallo--lactamase activity and stability. Biochemistry.
495
36:11534-11541.
496
17. Hernandez-Valladares, M., M. Kiefer, U. Heinz, R. Paul Soto, W. Meyer-Klaucke, H.
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Friederich Nolting, M. Zeppezauer, M. Galleni, J.M. Frère, G.M. Rossolini, G. Amicosante
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and H.W. Adolph. 2000. Kinetic and spectroscopic characterization of native and metal-
499
substituted -lactamase from Aeromonas hydrophila AE036. FEBS Lett. 467:221-225.
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18. Hooft, R.W.W., G. Vriend, C. Sander, E.E. and Abola. 1996. Errors in protein structures.
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Nature. 381:272-272.
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19. Horsfall, L.E., G. Garau, B.M. Liénard, O. Dideberg, C.J. Schofield, J.M. Frère, and M.
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Galleni. 2007 Competitive inhibitors of the CphA metallo--lactamase from Aeromonas
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hydrophila. Antimicrob. Agents Chemother. 51:2136-2142.
505
20. Kabsch, W. 1993. Automatic processing of rotation diffraction data from crystals of initially
506
unknown symmetry and cell constants. J. Appl. Cryst. 26:795-800.
507
21. Laskowski, R. A., M.W. MacArthur, D.S. Moss, and J.M. Thornton. 1993. PROCHECK:
508
a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26:283-291.
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22. Liénard, B.M.R., G. Garau, L. Horsfall, A.I. Karsisiotis, C. Damblon, P. Lassaux, C.
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Papamicael, G.C.K. Roberts, M. Galleni, O. Dideberg, J.M. Frère, and C.J. Schofield. 2008.
511
Structural basis for the broad-spectrum inhibition of metallo--lactamases by thiols. Org. Biomol.
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Chem. 6:2282-2294.
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23. Llarull, L.I., M.F. Tioni and A.J. Vila. 2008. Metal content and localization during turnover
514
in Bacillus cereus metallo--lactamase. J. Am. Chem. Soc. 130:15842-15851.
25
515
24. Morán-Barrio, J., J.M. González, M.N. Lisa, A.L. Costello, M. Dal Peraro, P. Carloni,
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B. Bennett, D.L. Tierney, A.S. Limansky, A.M. Viale, and A.J. Vila. 2007. The metallo--
517
lactamase GOB is a mono-Zn(II) enzyme with a novel active site. J. Biol. Chem. 282:18286-
518
18293.
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25. Murphy, T.A., L.E. Catto, S.E. Halford, A.T. Hadfield, W. Minor, T.R. Walsh, and J.
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Spencer. 2006. Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into
521
variable zinc affinity of metallo--lactamases. J.Mol.Biol. 357:890-903.
522
26. Murshudov, G.N., A.A. Vagin, and E.J. Dodson. 1997. Refinement of macromolecular
523
structures by the maximum-likelihood method. Acta Crystallogr. D53:240-255.
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27. Painter, J., E.A. and Merritt. 2006. Optimal description of a protein structure in terms of
525
multiple groups undergoing TLS motion. Acta Crystallogr. D62:439-450.
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28. Perrakis, A., R. Morris, and V.S. Lamzin. 1999. Automated protein model building
527
combined with iterative structure refinement. Nat Struct Biol. 6(5):458-463.
528
29. Segatore, B., O. Massida, G. Satta, D. Setacci, and G. Amicosante. 1993. High specificity
529
of cphA-encoded metallo--lactamase from Aeromonas hydrophila AE036 for carbapenems and
530
its contribution to -lactam resistance. Antimicrobial Agents and Chemother. 37:1324-1328.
531
30. Sharma, N.P., C. Hajdin, S. Chandrasekar, B. Bennett, K.W. Yang, and M.W. Crowder.
532
2006. Mechanistic studies on the mononuclear ZnII-containing metallo--lactamase ImiS from
533
Aeromonas sobria. Biochemistry. 45:10729-10738.
534
31. Tioni, M.F., L.I. Llarull, A.A. Poeylaut-Palena, M.A. Marti, M. Saggu, G.R. Periyannan,
535
E.G. Mata, B. Bennett, D.H. Murgida, and A.J. Vila. 2008. Trapping and characterization of a
536
reaction intermediate in carbapenem hydrolysis by Bacillus cereus metallo--lactamase. J. Am.
537
Chem. Soc. 130:15852-15863.
26
538
32. The CCP4. 1994. CCP4 suite: programs for protein crystallography. Acta Crystallog. Sect.
539
D. 50:760-763.
540
33. Ullah, J.H., T.R. Walsh, I.A. Taylor, D.C. Emery, C.S. Verma, S.J. Gamblin, and J.
541
Spencer. 1998. The crystal structure of the L1 metallo--lactamase from Stenotrophomonas
542
maltophilia at 1.7 Å resolution. J.Mol.Biol. 284:125-136.
543
34. Vanhove, M., M. Zakhem, B. Devreese, N. Franceschini, C. Anne, C. Bebrone, G.
544
Amicosante, G.M. Rossolini, J. Van Beeumen., J.M. Frère, and M. Galleni. 2003. Role of
545
Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo--lactamase.
546
Cell Mol Life Sci. 60:2501-2509.
547
35. Wommer, S., S. Rival, U. Heinz, M. Galleni, J.M. Frère, N. Franceschini, G.
548
Amicosante, B; Rasmussen, R. Bauer, and H.W. Adolph. 2002. Substrate-activated zinc
549
binding of metallo- -lactamases: physiological importance of mononuclear enzymes. J. Biol.
550
Chem. 277:24142-24147.
551
36. Xu, D., D. Xie, and H. Guo. 2006. Catalytic mechanism of class B2 metallo--lactamase. J.
552
Biol. Chem. 281:8740-8747.
553
37. Yanchak, M.P., Taylor, R.A. and M.W. Crowder. 2000. Mutational analysis of metallo--
554
lactamase CcrA from Bacteroides fragilis. Biochemistry. 39:11330-11339.
555
27
556
Table 1. 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 MLs
His116-His118-His196
Asp120-Cys221-His263
B2 MLs
His118-His196
Asp120-Cys221-His263
B3 MLs
His116-His118-His196
Asp120-His121-His263
B3 ML 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