Asp-120 Locates Zn2 for Optimal Metallo-β-lactamase Activity
2007; Elsevier BV; Volume: 282; Issue: 25 Linguagem: Inglês
10.1074/jbc.m700742200
ISSN1083-351X
AutoresLeticia I. Llarrull, Stella M. Fabiane, Jason M. Kowalski, Brian Bennett, Brian J. Sutton, Alejandro J. Vila,
Tópico(s)Tuberculosis Research and Epidemiology
ResumoMetallo-β-lactamases are zinc-dependent hydrolases that inactivate β-lactam antibiotics, rendering bacteria resistant to them. Asp-120 is fully conserved in all metallo-β-lactamases and is central to catalysis. Several roles have been proposed for Asp-120, but so far there is no agreed consensus. We generated four site-specifically substituted variants of the enzyme BcII from Bacillus cereus as follows: D120N, D120E, D120Q, and D120S. Replacement of Asp-120 by other residues with very different metal ligating capabilities severely impairs the lactamase activity without abolishing metal binding to the mutated site. A kinetic study of these mutants indicates that Asp-120 is not the proton donor, nor does it play an essential role in nucleophilic activation. Spectroscopic and crystallographic analysis of D120S BcII, the least active mutant bearing the weakest metal ligand in the series, reveals that this enzyme is able to accommodate a dinuclear center and that perturbations in the active site are limited to the Zn2 site. It is proposed that the role of Asp-120 is to act as a strong Zn2 ligand, locating this ion optimally for substrate binding, stabilization of the development of a partial negative charge in the β-lactam nitrogen, and protonation of this atom by a zinc-bound water molecule. Metallo-β-lactamases are zinc-dependent hydrolases that inactivate β-lactam antibiotics, rendering bacteria resistant to them. Asp-120 is fully conserved in all metallo-β-lactamases and is central to catalysis. Several roles have been proposed for Asp-120, but so far there is no agreed consensus. We generated four site-specifically substituted variants of the enzyme BcII from Bacillus cereus as follows: D120N, D120E, D120Q, and D120S. Replacement of Asp-120 by other residues with very different metal ligating capabilities severely impairs the lactamase activity without abolishing metal binding to the mutated site. A kinetic study of these mutants indicates that Asp-120 is not the proton donor, nor does it play an essential role in nucleophilic activation. Spectroscopic and crystallographic analysis of D120S BcII, the least active mutant bearing the weakest metal ligand in the series, reveals that this enzyme is able to accommodate a dinuclear center and that perturbations in the active site are limited to the Zn2 site. It is proposed that the role of Asp-120 is to act as a strong Zn2 ligand, locating this ion optimally for substrate binding, stabilization of the development of a partial negative charge in the β-lactam nitrogen, and protonation of this atom by a zinc-bound water molecule. β-Lactamases are hydrolytic enzymes produced by bacteria as a mechanism of resistance to β-lactam antibiotics (1Fisher J.F. Meroueh S.O. Mobashery S. Chem. Rev. 2005; 105: 395-424Crossref PubMed Scopus (766) Google Scholar, 2Wilke M.S. Lovering A.L. Strynadka N.C. Curr. Opin. Microbiol. 2005; 8: 525-533Crossref PubMed Scopus (286) Google Scholar). These enzymes are capable of catalyzing the scission of the amide bond of the β-lactam ring characteristic of this class of antibiotics, rendering them ineffective toward their targets. They can be broadly divided into serine-β-lactamases and metallo-β-lactamases, based on their active sites and catalytic mechanisms. β-Lactam hydrolysis takes place by a nucleophilic attack to the carbonyl group, and a subsequent C-N cleavage, usually aided by protonation of the bridging nitrogen atom (3Page M.I. Laws A.P. J. Chem. Soc. Chem. Commun. 1998; 1998: 1609-1617Crossref Scopus (142) Google Scholar) (Scheme 1). In the case of serine-β-lactamases, the reaction proceeds through formation of a covalent intermediate with the nucleophilic Ser-70 (4Strynadka N.C. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N. Nature. 1992; 359: 700-705Crossref PubMed Scopus (533) Google Scholar). Based on this mechanistic feature, clinically useful inhibitors for serine-β-lactamases have been designed, such as clavulanic acid and tazobactam, that give rise to irreversible inhibition by formation of a covalent adduct with the enzyme (5Sulton D. Pagan-Roderiguez D. Zhou X. Liu Y. Hujer A.M. Bethel C.R. Helfand M.S. Thomson J.M. Anderson V.E. Buynak J.D. Ng L.M. Bonomo R.A. J. Biol. Chem. 2005; 280: 35528-35536Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In contrast, MβL 4The abbreviations used are: MβL, metallo-β-lactamase; SKIE, solvent kinetic isotope effect; CT, charge transfer; WT, wild type. -mediated catalysis does not proceed through such an intermediate, thus rendering these inhibitors ineffective (6Crowder M.W. Spencer J. Vila A.J. Acc. Chem. Res. 2006; 39: 721-728Crossref PubMed Scopus (330) Google Scholar). The spread of plasmid-encoded MβL genes among opportunistic and pathogenic bacteria, together with the lack of clinically useful inhibitors, is becoming a serious and yet unsolved clinical problem (7Walsh T.R. Toleman M.A. Poirel L. Nordmann P. Clin. Microbiol. Rev. 2005; 18: 306-325Crossref PubMed Scopus (1226) Google Scholar). The elucidation of the catalytic mechanisms employed by MβLs to hydrolyze β-lactam antibiotics is a prerequisite for rational inhibitor design. The activity of MβLs is dependent on the presence of either one or two Zn(II) ions in their active sites. MβLs have been classified into three subgroups (B1, B2, and B3), based on amino acid sequence similarity, substrate profile, and structural properties (8Garau G. Di Guilmi A.M. Hall B.G. Antimicrob. Agents Chemother. 2005; 49: 2778-2784Crossref PubMed Scopus (65) Google Scholar). The diversity of these subgroups, exemplified by the vastly different efficacies of nonclinical inhibitors toward MβLs, led to the prediction that finding a single inhibitor for all metallo-β-lactamases may not be possible. The search for any common mechanistic feature of MβLs is therefore a high priority. The first crystal structure solved for an MβL was that of BcII from Bacillus cereus, which revealed one Zn(II) ion bound to three His residues (His-116, His-118, and His-196) and a H2O molecule, in the so-called Zn1 or 3H site (9Carfi A. Pares S. Duee E. Galleni M. Duez C. Frère J.M. Dideberg O. EMBO J. 1995; 14: 4914-4921Crossref PubMed Scopus (408) Google Scholar). Subsequent structures of BcII (10Fabiane S.M. Sohi M.K. Wan T. Payne D.J. Bateson J.H. Mitchell T. Sutton B.J. Biochemistry. 1998; 37: 12404-12411Crossref PubMed Scopus (217) Google Scholar) and other B1 MβLs (11Concha N. Rasmussen B.A. Bush K. Herzberg O. Structure. 1996; 4: 823-836Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 12Garcia-Saez I. Hopkins J. Papamicael C. Franceschini N. Amicosante G. Rossolini G.M. Galleni M. Frere J.M. Dideberg O. J. Biol. Chem. 2003; 278: 23868-23873Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 13Toney J.H. Hammond G.G. Fitzgerald P.M. Sharma N. Balkovec J.M. Rouen G.P. Olson S.H. Hammond M.L. Greenlee M.L. Gao Y.D. J. Biol. Chem. 2001; 276: 31913-31918Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) revealed a dinuclear metal center containing the tetrahedral 3H site and an additional trigonal bipyramidal Zn(II) site (the Zn2 or DCH site), where the metal ion is coordinated to Asp-120, Cys-221, His-263, a bridging H2O/OH–, and an additional water molecule (Fig. 1). An analogous binding site in B3 enzymes is provided by Asp-120, His-121, and His-263 (DHH site) (14Garcia-Saez I. Mercuri P.S. Papamicael C. Kahn R. Frere J.M. Galleni M. Rossolini G.M. Dideberg O. J. Mol. Biol. 2003; 325: 651-660Crossref PubMed Scopus (116) Google Scholar, 15Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spencer J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (299) Google Scholar). B2 enzymes, however, are active as mononuclear enzymes, with the only Zn(II) ion located in the DCH site (16Garau G. Bebrone C. Anne C. Galleni M. Frere J.M. Dideberg O. J. Mol. Biol. 2005; 345: 785-795Crossref PubMed Scopus (190) Google Scholar). Despite these differences, Asp-120 is fully conserved in all metallo-β-lactamases identified so far. Substitution of Asp-120 in B1 and B3 MβLs have shown that this residue is central to catalysis (17Seny D. Prosperi-Meys C. Bebrone C. Rossolini G.M. Page M.I. Noel P. Frere J.M. Galleni M. Biochem. J. 2002; 363: 687-696Crossref PubMed Google Scholar, 18Yanchak M.P. Taylor R.A. Crowder M.W. Biochemistry. 2000; 39: 11330-11339Crossref PubMed Scopus (83) Google Scholar, 19Garrity J.D. 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Despite all the efforts aimed at determining the role of Asp-120 in the hydrolysis of β-lactam antibiotics, there is no agreed consensus. To provide further evidence regarding the role of Asp-120, we generated four site-specifically substituted variants of the enzyme BcII from B. cereus. Asp-120 was replaced by an asparagine, a glutamic acid, a glutamine, and a serine to create the BcII mutant proteins D120N, D120E, D120Q, and D120S, respectively. Based on a series of biochemical, spectroscopic, and crystallographic studies, here we propose that the principal role of Asp-120 in MβLs is defining the position of the Zn2 ion, which is crucial for stabilizing the development of a negative charge on the β-lactam nitrogen atom, providing the water molecule that protonates this nitrogen, and binding the substrate. All chemicals were of the highest quality available. Escherichia coli BL21(DE3)pLysS′ cells (Stratagene, CA) were employed for protein production. E. coli JM109 cells (Stratagene, CA) were employed for transformation with plasmid DNA and ligation mixtures. Luria-Bertani medium (Sigma) was used as growth media for all bacterial strains. DNA preparation and related techniques were performed according to standard protocols (29Sambrook J. Fitsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor, NY1989Google Scholar). Plasmid DNA was isolated using the Wizard Plus SV minipreps kit (Promega). DNA was extracted from agarose gels using QIAEX II kit (Qiagen) or GFX columns (Amersham Biosciences). A preparation of pETβLII plasmid DNA (30Orellano E.G. Girardini J.E. Cricco J.A. Ceccarelli E.A. Vila A.J. Biochemistry. 1998; 37: 10173-10180Crossref PubMed Scopus (115) Google Scholar) was digested with BamHI and PstI and subcloned into a vector pBluescript II KS(–) previously digested with the same restriction endonucleases, to obtain the plasmid KS-NH3, which contains the DNA fragment coding for the NH3-terminal half of BcII (31Cricco J.A. Role of a Cys Residue in the Structure and Function of Metallo-beta-Lactamases, University of Rosario, Rosario, Argentina2002Google Scholar). This plasmid was used as the DNA template for the PCR-based mutagenesis protocol. Site-directed mutagenesis was performed using the megaprimer PCR protocol (32Barik S. Trower M.K. Site-directed Mutagenesis in Vitro by Megaprimer PCR. Humana Press Inc., Totowa, NJ1996: 203-216Google Scholar). The first PCR was carried out using the plasmid KS-NH3 as the template DNA, the ks-reverse primer (5′-TCACACAggAAACAgCTATgAC-3′), and the corresponding mutagenic primer: D120_N_SphI (5′-CACATgCgCATgCTAATCgAATTggCgg-3′) for D120N, D120_S_SphI (5′-CACATgCgCATgCTAgTCgAATTggCgg-3′) for D120S, D120_E_SphI (5′-CACATgCgCATgCTgAACgAATTggCgg-3′) for D120E, and D120_Q_SphI (5′-CACATgCgCATgCTCAACgAATTggCgg-3′) for D120Q. The mutagenic primers were designed to introduce a recognition site for the restriction endonuclease SphI, through the introduction of a silent mutation, using the software Primer Tailoring (33Llarrull L.I. Calcaterra N.B. Biocell. 2001; 25: 62Google Scholar). Boldface underlined letters indicate the silent mutation, and boldface italic underlined letters indicate the nonsilent mutations. A 100-μl reaction mixture, containing 0.2 mm of each dNTP, 0.5 pmol/μl of each primer, 1 mm MgSO4, 100 ng of template DNA (KS-NH3), 10 μl of the 10× buffer provided with the polymerase (New England Biolabs), and 2 units of Vent DNA polymerase (New England Biolabs), was used in the PCR, using a GeneAmp® PCR System 2400 equipment (PerkinElmer Life Sciences). The following cycles were employed in the first PCR: 1) 1 cycle of 3 min at 94 °C; 2) pause for the addition of the DNA polymerase; 3) 30 cycles of 2 min at 94 °C, 3 min at 48 °C, and 2 min at 72 °C; and 4) 1 cycle of 3 min at 72 °C. The PCR mixture was resolved in a 2% agarose gel containing ethidium bromide, and the 210-bp PCR product (the megaprimer) was recovered from the excised agarose fragment using the QIAEX II kit (Qiagen). The second PCR was carried out using the same template DNA, the megaprimer that codes for the desired mutation, and the ks-forward primer (5′-CgCCAgggTTTTCCCAgTCACgAC-3′). All the megaprimer recovered from the first PCR was used in the second round of amplification, in a 50-μl reaction mixture, containing 0.2 mm of each dNTP, 0.5 pmol/μl of the ks-forward primer, 1 mm MgSO4, 100 ng of template DNA (KS-NH3), 5 μl of the 10× buffer provided with the polymerase (New England Biolabs Inc.), and 1.2 units of Vent DNA polymerase (New England Biolabs Inc.). The following cycles were employed in the second PCR: 1) 1 cycle of 5 min at 94 °C; 2) pause for the addition of the DNA polymerase; 3) 35 cycles of 2 min at 94 °C, 2 min at 55 °C, and 2 min at 72 °C; and 4) 1 cycle of 4 min at 72 °C. The amplification was corroborated by gel electrophoresis, and the 579-bp purified PCR product was digested with the restriction endonucleases BamHI and PstI and cloned into pBluescript II KS(–), previously digested with the same endonucleases. After transformation of E. coli JM109 cells with the ligation mixture, the presence of the mutated DNA sequences in the plasmid DNA preparations from selected clones was first corroborated by digestion of the DNA with the restriction endonuclease SphI, whose recognition site was introduced by the mutagenic primers. Afterward, the sequences were confirmed by DNA sequencing (University of Maine Sequencing Facility). The genes that code for the Asp-120 mutants of the metallo-β-lactamase BcII were reconstructed by cloning the DNA fragment coding for the mutagenized NH3-half of BcII, digested with the enzymes BamHI and PstI, and the DNA fragment coding for the wild-type COOH-half of BcII, digested with PstI and HindIII, into the plasmid pET-TERM (31Cricco J.A. Role of a Cys Residue in the Structure and Function of Metallo-beta-Lactamases, University of Rosario, Rosario, Argentina2002Google Scholar) previously digested with the restriction endonucleases BamHI and HindIII. The expression vector pET-TERM allows expression of the protein of interest as an amino-terminal fusion to the enzyme glutathione S-transferase from Schistosoma japonicum, under control of the T7 promoter, and presents the termination sequence from the BcII gene. The DNA fragment that codes for the COOH-terminal half of BcII was purified, after digestion with PstI and HindIII, from the plasmid KS-CT2 (31Cricco J.A. Role of a Cys Residue in the Structure and Function of Metallo-beta-Lactamases, University of Rosario, Rosario, Argentina2002Google Scholar). Wild-type BcII and Asp-120 mutants were expressed in E. coli BL21(DE3)pLysS′ cells as fusion proteins with glutathione S-transferase, purified, and quantified as follows. Typically, the cell pellet obtained from 50 ml of a saturated culture in LB medium, supplemented with 150 μg/ml ampicillin and 35 μg/ml chloramphenicol, was resuspended in fresh medium and used to inoculate 1-liter of LB medium supplemented with 150 μg/ml ampicillin and 35 μg/ml chloramphenicol, in a 5-liter Erlenmeyer flask. Cells were grown for 2 h at 37 °C until the A600 = 1 was reached. The expression of fusion protein was induced by addition of 10 g of lactose, and the culture was further incubated at 37 °C for an additional 4-h period. All subsequent purification steps were performed at 4 °C. E. coli cells were lysed by sonication in lysis buffer (16 mm Na2HPO4, 4 mm NaH2PO4, and 150 mm NaCl, pH 7, with 3.3 μg/ml DNase, 5 mm MgCl2, and 1 mm phenylmethylsulfonyl fluoride), and cell debris was separated by ultracentrifugation. The GST-BcII fusion protein was purified by affinity chromatography using a glutathione-Sepharose resin (Amersham Biosciences) as reported previously (30Orellano E.G. Girardini J.E. Cricco J.A. Ceccarelli E.A. Vila A.J. Biochemistry. 1998; 37: 10173-10180Crossref PubMed Scopus (115) Google Scholar). The pure fractions were treated with bovine plasma thrombin (Sigma) at a 1:100 ratio in 150 mm NaCl, 2.5 mm CaCl2 at 26 °C during 2 h. BcII was purified from the proteolysis mixture by ion-exchange chromatography on Sephadex CM-50 (Amersham Biosciences) as reported previously (30Orellano E.G. Girardini J.E. Cricco J.A. Ceccarelli E.A. Vila A.J. Biochemistry. 1998; 37: 10173-10180Crossref PubMed Scopus (115) Google Scholar). Protein samples were dialyzed against >100 volumes of 10 mm HEPES, pH 7.5, 0.2 m NaCl. Purity of the enzyme preparations was checked by SDS-PAGE. Protein concentration was measured spectrophotometrically using ϵ280 = 30,500 m–1·cm–1 (34Paul-Soto R. Bauer R. Frère J.M. Galleni M. Meyer-Klaucke W. Nolting H. Rossolini G.M. de Seny D. Hernández Valladares M. Zeppezauer M. Adolph H.W. J. Biol. Chem. 1999; 274: 13242-13249Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The metal content in the samples of wild-type BcII and Asp-120 mutants was determined under denaturing conditions using the colorimetric metal chelator 4-(2-pyridylazo)resorcinol, as described by Fast et al. (35Fast W. Wang Z. Benkovic S.J. Biochemistry. 2001; 40: 1640-1650Crossref PubMed Scopus (76) Google Scholar). The kinetic parameters for the hydrolysis of different β-lactam antibiotics catalyzed by wild-type BcII and Asp-120 mutants under steady-state conditions were obtained by determination of the initial rate of reaction at different substrate concentrations. Substrate concentrations were calculated based on the following molar absorptivities: penicillin G, Δϵ235 = –775 m–1·cm–1; cefotaxime, Δϵ260 = –7,500 m–1·cm–1; nitrocefin, Δϵ485 = 17,420 m–1·cm–1; imipenem, Δϵ300 = –9,000 m–1·cm–1. The plots of the dependence of the initial rates on substrate concentration were fitted to the Michaelis and Menten equation, using SigmaPlot 8.0. Reactions were carried out in 10 mm HEPES, pH 7.5, 200 mm NaCl, 20 μm ZnSO4, and 0.05 mg/ml bovine serum albumin at 30 °C. Absorbance changes upon substrate hydrolysis were measured in a Jasco V-550 spectrophotometer, and the temperature was kept constant by means of a Polyscience digital circulator connected to the cell holder in the spectrophotometer. The kinetic parameters obtained for the hydrolysis of different β-lactam antibiotics catalyzed by wild-type BcII and Asp-120 mutants in H2O, under steady-state conditions, were compared with the kinetic parameters obtained for the same reaction carried out in D2O. The initial rate of hydrolysis at different substrate concentrations was measured in 10 mm HEPES, pD 7.5 (pH 7.1), 200 mm NaCl, 20 μm ZnSO4, and 0.05 mg/ml bovine serum albumin at 30 °C. The dependence of the initial rates on substrate concentration was fitted to the Michaelis-Menten equation, using SigmaPlot 8.0. The deuterated reaction medium was kept at 30 °C under a N2 gas atmosphere, prior to reaction. The hydrolysis of the antibiotics was registered in open cells, during less than 5 min, to avoid a significant water uptake from the environment. All buffer solutions used to prepare the apoenzymes were treated by extensive stirring with Chelex 100 (Sigma). Apoprotein samples were prepared by dialysis of the purified holoprotein (∼200 μm) against two changes of >100 volumes of 10 mm HEPES, pH 7.5, 0.2 m NaCl, 20 mm EDTA over a 12-h period under stirring (36Paul-Soto R. Hernandez Valladares M. Galleni M. Bauer R. Zeppezauer M. Frere J.M. Adolph H.W. FEBS Lett. 1998; 438: 137-140Crossref PubMed Scopus (64) Google Scholar). EDTA was removed from the resulting apoenzyme solution by three dialysis steps against >100 volumes of 10 mm HEPES, pH 7.5, 1 m NaCl, Chelex 100, and three dialysis steps against >100 volumes of 10 mm HEPES, pH 7.5, 0.2 m NaCl, Chelex 100 (36Paul-Soto R. Hernandez Valladares M. Galleni M. Bauer R. Zeppezauer M. Frere J.M. Adolph H.W. FEBS Lett. 1998; 438: 137-140Crossref PubMed Scopus (64) Google Scholar). For the preparation of apoprotein samples used in EPR experiments, the three last dialysis steps were replaced by one dialysis step against >100 volumes of 10 mm HEPES, pH 7.5, 0.2 m NaCl, Chelex 100 and finally two dialysis steps against >100 volumes of 100 mm HEPES, pH 7.5, 0.2 m NaCl, Chelex 100. All dialysis steps were carried out at 4 °C. A solution of 200–300 μm apoprotein in 10 mm HEPES, pH 7.5, 0.2 m NaCl was titrated with a 10.6 mm CoSO4 stock solution prepared in 10 mm HEPES, pH 7.5, 0.2 m NaCl. For the UV-visible titration, which was done in parallel with the EPR titration, a solution of 1.2 mm apo-BcII D120S in 100 mm HEPES, pH 7.5, 0.2 m NaCl was titrated with a 6 mm CoSO4 stock solution prepared in 100 mm HEPES, pH 7.5, 0.2 m NaCl. The equivalents of bound Co(II) were calculated as the ratio between the concentration of Co(II) in the sample and the concentration of protein capable of binding metal, which was calculated by multiplying the concentration of protein determined by absorbance at 280 nm by the factor n/2, where n is the Zn(II) content of the purified proteins. The spectra were recorded at room temperature in a UV-visible Jasco V-550 spectrophotometer, and difference spectra were obtained by subtracting the spectrum of the corresponding apoprotein. EPR spectroscopy was performed at 13 K, 2 milliwatts, and 9.63 GHz (Bmicrowave ⊥ Bstatic) using a Bruker Elexsys E500 spectrometer equipped with an ER 4116 DM TE012/TE102 dual mode X-band cavity and an Oxford Instruments ESR-900 helium flow cryostat. The apoprotein (1.2 mm), in 100 mm HEPES, pH 7.5, 0.2 m NaCl, was titrated stepwise with a 6 mm CoSO4 stock solution prepared in the same buffer. The Co(II)-containing solution was rapidly mixed with the sample in the EPR tube by manual flicking (37Bennett B. Benson N. McEwan A.G. Bray R.C. Eur. J. Biochem. 1994; 225: 321-331Crossref PubMed Scopus (98) Google Scholar) and frozen in liquid nitrogen. EPR samples for successive additions of Co(II) were quickly thawed (∼5 s) from 77 to 293 K by agitating the sample tube in water. Crystallization and Data Collection—D120S was crystallized using the vapor diffusion method. 1 μl of the protein solution, at 4.7 mg/ml, was mixed with 1 μl of reservoir solution, containing 17–19% PEG-3350, 0.1 m sodium cacodylate, pH 5.0–5.5, 0.1 m sodium tartrate. Large single crystals grew within a few weeks. These were transferred to a solution supplemented with 25% glycerol and cryocooled by plunging them into liquid nitrogen. The crystals diffract to 1.95 Å resolution, and a data set was collected at CCLRC Daresbury Synchrotron Radiation Source, station 14.2, at a wavelength of 0.9853Å. The space group was C2, with unit cell parameters a = 53.39 Å, b = 61.97Å, c = 69.57Å, and β = 93.28°. The data were reduced and scaled using MOSFLM (38Leslie A.G.W. Joint CCP4 and ESF-EAMBC Newsl. on Protein Crystallogr. 1992; 26: 27-33Google Scholar) and SCALA as implemented in the CCP4 package (39Collaborative Computational Project Number 4.Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19825) Google Scholar). Data processing statistics are given on Table 2. 5% of the reflections, chosen randomly, were assigned for the Rfree calculations.TABLE 2Crystallographic data collection and refinement statisticsDiffraction data Resolution (Å)1.95 Unit cella = 53.39 Å, b = 61.97 Å, c = 69.57 Å, β = 93.28° No. of unique reflections16,580 Completeness (%) (2.00 to 1.95 Å)99.9 (99.9) Rmerge (%)7.5 (31.7) I/σ(I)7.6 (2.3)Refinement Resolution limits (Å)31.0 to 1.95 R (%, 95% reflections)14.6 Rfree (%, 5% reflections)22.1 DeviationsBond lengths (Å)0.013Bond angles (°)1.366 Mean B factorsMain-chain atoms (Å2)25.44Side-chain atoms (Å2)26.98Zinc atoms (Å2)22.02Hetero atoms (Å2)19.47Water atoms (Å2)37.27Ramachandran plot Favored (%, number)92.6, 176 Additional allowed (%, number)6.8, 13 Generously allowed (%, number)0, 0 Disallowed (%, number)0.5, 1 Open table in a new tab Structure Determination and Refinement—The structure was determined by molecular replacement using CNS (40Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16991) Google Scholar). The crystal structure of the wild-type BcII (Protein Data Bank code 3BC2) was used as a search model, stripped of all nonconvalently bound atoms. One clear solution was found, and rigid body refinement yielded R-factor of 30.9% and Rfree was 31.6%. The structure was refined using CNS during two rounds, and finished using Refmac5 from the CCP4 package. Model building was performed using Quanta (Accelrys) and Coot (41Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23864) Google Scholar). The TLS groups were chosen using TLSMD (42Painter J. Merritt E.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 439-450Crossref PubMed Scopus (1111) Google Scholar), run from the TLSMD server (43Painter J. Merritt E.A. J. Appl. Crystallogr. 2006; 39: 109-111Crossref Scopus (655) Google Scholar). The final Rfree was 22.1%, R-factor for 95% of reflections was 14.6%. It was clear that some of the noncovalently bound atoms (including the Zn(II) ion in position Zn2) were not fully occupied. At 1.95 Å resolution, it was not possible to refine their occupancy and temperature factor simultaneously, and so their occupancies were fixed at 0.5. The final model contains 218 residues, two zinc ions, four glycerol molecules, and 283 waters. Asp-84, as is the case in other structures of the same protein, is in the disallowed region of the Ramachandran plot. The geometry of the model is good as checked by SFCHECK (44Vaguine A.A. Richelle J. Wodak S.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 191-205Crossref PubMed Scopus (862) Google Scholar), PROCHECK (45Laskowski R.A. Mac Arthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), MolProbity (46Lovell S.C. Davis I.W. Arendall III, W.B. de Bakker P.I. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Proteins. 2003; 50: 437-450Crossref PubMed Scopus (3914) Google Scholar), and Coot (41Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23864) Google Scholar). Biochemical and Enzymatic Characterization of BcII Asp-120 Mutants—To probe the role of Asp-120 in MβLs, we designed the following point mutants: D120S, D120N, D120E,
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