A recombinant form of Escherichia coli DdlB (EcDdlB) has been prepared and cocrystallized with ADP and D-alanyl-D-alanine to represent the ternary complex of EcDdlB. Furthermore, EcDdlB has been cocrystallized under the same conditions with the ligands ATP and D-alanyl-D-alanine, representing the product-inhibited complex. The crystals belonged to space group P212121, with unit-cell parameters a = 53.0, b = 97.6, c = 109.5 Å and a = 51.2, b = 97.8, c = 110.1 Å, respectively, and both contained two molecules in the asymmetric unit. Complete data sets were collected to 1.5 and 1.4 Å resolution, respectively, from single crystals under cryogenic conditions using synchrotron radiation

Batson, Sarah

Fülöp, Vilmos

Rea, Dean

Roper, David I.

English

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 University of Warwick institutional repository: http://go.warwick.ac.uk/wrap  This paper is made available online in accordance with publisher policies. Please scroll down to view the document itself. Please refer to the repository record for this item and our policy information available from the repository home page for further information.  To see the final version of this paper please visit the publisher’s website. Access to the published version may require a subscription. Author(s):  Sarah Batson, Dean Rea,Vilmos Fülöpa and David I. Roper Article Title:  Crystallization and preliminary X-ray analysis of a D-alanyl-D-alanine ligase (EcDdlB) from Escherichia coli Year of publication: 2010 Link to published article:  http://dx.doi.org/10.1107/S1744309110003970 Publisher statement:  None        crystallization communicationsActa Cryst. (2010). F66, 405–408 doi:10.1107/S1744309110003970 405Acta Crystallographica Section FStructural Biologyand CrystallizationCommunicationsISSN 1744-3091Crystallization and preliminary X-ray analysis of aD-alanyl-D-alanine ligase (EcDdlB) from EscherichiacoliSarah Batson, Dean Rea, VilmosFu¨lo¨p and David I. Roper*Department of Biological Sciences, University ofWarwick, Coventry CV4 7AL, EnglandCorrespondence e-mail:david.roper@warwick.ac.ukReceived 26 November 2009Accepted 1 February 2010A recombinant form of Escherichia coli DdlB (EcDdlB) has been preparedand cocrystallized with ADP and d-alanyl-d-alanine to represent the ternarycomplex of EcDdlB. Furthermore, EcDdlB has been cocrystallized under thesame conditions with the ligands ATP and d-alanyl-d-alanine, representing theproduct-inhibited complex. The crystals belonged to space group P212121, withunit-cell parameters a = 53.0, b = 97.6, c = 109.5 A˚ and a = 51.2, b = 97.8,c = 110.1 A˚, respectively, and both contained two molecules in the asymmetricunit. Complete data sets were collected to 1.5 and 1.4 A˚ resolution, respectively,from single crystals under cryogenic conditions using synchrotron radiation.1. IntroductionPeptidoglycan is a specific dynamic structure which constitutes a vitalcomponent of the bacterial cell wall (Vollmer et al., 2008). The bio-synthesis of peptidoglycan occurs in three distinct phases at differentlocations and stages of the cell cycle. The initial cytoplasmic stagesequentially assembles the peptidoglycan intermediates prior to theirtranslocation across the cell membrane. At the bacterial exoplasmicsurface subsequent transpeptidation and transglycosylation reactionsoccur to produce mature cross-linked chains of peptidoglycan.d-Alanyl-d-alanine ligase (Ddl) is a cytoplasmic enzyme specific topeptidoglycan synthesis; it provides the d-alanyl-d-alanine dipeptidesubstrate for MurF in the initial cytoplasmic phase of biosynthesis(Neuhaus, 1962). The d-alanyl-d-alanine dipeptide is a central com-ponent of the biosynthetic pathway and is produced by Ddl in anATP-dependent reaction mechanism which proceeds in two steps(Parsons et al., 1988; Mullins et al., 1990). Although d-cycloserine haslong been known to inhibit Ddls and alanine racemase, problemsarising from the toxicity of the drug mean that its administration isrestricted (Neuhaus & Lynch, 1962) apart from the treatment ofMycobacterium tuberculosis infections. Nevertheless, Ddls present anattractive target for chemotherapeutic intervention because of theiressential and universal role in bacterial cell-wall formation.The enterococcal homologue of EcDdlB, d-alanyl-d-alanine ligase(VanA), functions in the mechanism of vancomycin resistance andalso represents a novel target in the reversal of vancomycin resistance(Bugg et al., 1991). Therefore, the study of these enzymes is of primeinterest in the search for new antimicrobials, particularly by rationaldrug-design methods. In order to elucidate the structural andchemical basis of enzyme activity, the crystal structures of Ddl fromEscherichia coli (Fan et al., 1994), Staphylococcus aureus (Liu et al.,2006), Thermus caldophilus (Lee et al., 2006),Helicobacter pylori (Wuet al., 2008) and T. thermophilus HB8 (Kitamura et al., 2009) havebeen reported. Additionally, the crystal structures of d-alanyl-d-lactate ligases from Enterococcus faecium (Roper et al., 2000) andLeuconostoc mesenteroides (Kuzin et al., 2000) have been reported.EcDdlB represents one of the two isoforms of Ddl that exist inE. coli and that have been kinetically characterized previously(Zawadzke et al., 1991). Currently, two wild-type structures of E. coliEcDdlB have been solved by X-ray crystallography in addition to amutant form, Y216F EcDdlB. The first of these represented the first# 2010 International Union of CrystallographyAll rights reservedstructure of a Ddl to be solved (Fan et al., 1994) and is in complexwith ADP and a phosphophosphinate transition-state analogue. Thestructure identified several active-site residues and formed the basisfor much of the subsequent work in the field of the Ddls (Shi &Walsh, 1995; Park et al., 1996; Lessard et al., 1999; Prevost et al., 2000).The subsequent structures of wild-type EcDdlB and Y216F EcDdlBwith phosphonate and phosphinate intermediates revealed no majorconformational changes in comparison to the first published structure(Fan et al., 1997): both are in complex with a transition-state inter-mediate and exist in closed enzyme conformations. Here, we reportthe crystallization and preliminary characterization of EcDdlB incomplex with ATP and d-alanyl-d-alanine and in complex with ADPand d-alanyl-d-alanine. d-Alanyl-d-alanine acts as a substrate for thereverse reaction of ADP/Pi-dependent phosphorylysis, which occursat a Vmax of less than 1% of the forward reaction (Mullins et al., 1990).EcDdlB has also previously been reported to display noncompetitiveproduct inhibition (with a reported Ki of 49 mM; Zawadzke et al.,1991). This suggests that the mode of binding of d-alanyl-d-alanine tothe active site is different as a substrate and as an inhibitor, formingthe rationale for cocrystallization studies of EcDdlB with ATP andd-alanyl-d-alanine. Furthermore, in contrast to previously reportedtransition-state mimic-containing EcDdlB structures, crystallographicstudies of EcDdlB containing ADP and d-alanyl-d-alanine have beenperformed in order to determine the true ternary complex structureof EcDdlB. Further structural information regarding EcDdlB maycontribute to a greater structural understanding of EcDdlB and maycontribute to the design of effective ligase inhibitors.2. Materials and methods2.1. Cloning, overproduction and purification of E. coli EcDdlBThe gene for EcDdlB was amplified by PCR and cloned into theNcoI and XhoI sites of pProEXa (Invitrogen) to produce an amino-terminally histidine-tagged protein; the tag was not removed forcrystallization. Freshly transformed E. coli BL21(DE3)Star-pRosetta cells harbouring pProEX::EcDdlB were grown in 1 l Luriabroth medium containing 50 mg ml1 ampicillin and 30 mg ml1chloramphenicol. The culture was grown at 310 K to an OD600 of 0.5,at which point isopropyl -d-1-thiogalactopyranoside was added to afinal concentration of 1 mM. After 3 h of induction at 310 K, cellswere harvested by centrifugation, resuspended in 30 ml buffer A(50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole) andsonicated. Cellular debris was removed by centrifugation at 20 000gfor 30 min and the supernatant was applied onto a 5 ml HisTrap FastFlow column (GE Healthcare) equilibrated with buffer A. EcDdlBwas eluted with a gradient of 10–250 mM imidazole over 60 ml.The fractions containing EcDdlB were pooled, concentrated with aCentripep-30 concentrator and applied onto a Superdex 200 columnequilibrated with 150 mM KCl, 50 mM HEPES pH 7.5 and 0.5 mMEDTA. EcDdlB was eluted with approximately 180 ml buffer. Thefractions containing recombinant EcDdlB were analysed by SDS–PAGE (Fig. 1), pooled and concentrated to 12 mg ml1 using aCentriprep-30 concentrator. Enzyme activity was determined using acontinuous spectrometric assay monitoring ADP release as describedpreviously (Daub et al., 1988).2.2. CrystallizationAll crystallization experiments were performed using vapourdiffusion. Initial crystallization was performed using the sitting-dropvapour-diffusion method. The protein concentration used for crys-tallization was 12 mg ml1 in 5 mM ATP or 5 mM ADP and 50 mMd-alanyl-d-alanine and a ratio of one volume of protein solution toone volume of precipitant solution was used throughout. Crystalswere obtained in conditions from a commercially available crystalscreen (Molecular Dimensions). Refinement of the Tris–HCl condi-tions from pH 8.5 to pH 8 using the hanging-drop vapour-diffusionmethod gave crystals of sufficient quality for data collection.2.3. Data collectionSingle crystals of the EcDdlB complexes were flash-cooled inliquid nitrogen using reservoir solution containing 30% glycerol as acryoprotectant. X-ray diffraction data were collected on beamline I04at Diamond (UK). Diffraction images were measured using an ADSCQuantum 315 CCD detector.3. Results and discussionThe E. coli EcDdlB protein was purified by immobilized metal-affinity chromatography and size-exclusion chromatography to yield10 mg purified protein from 1 l culture. SDS–PAGE analysis revealedthat the preparation was free of major and minor contaminatingspecies. The subunit molecular mass on SDS–PAGE appeared to beconsistent with the calculated molecular mass of 34 533 Da. Thecatalytic efficiency for d-alanyl-d-alanine synthesis was found to be477.4  0.7 min1 mM1, a value that is comparable to that obtainedin a previous kinetic analysis of EcDdlB (440 min1 mM1) in a non-histidine-tagged form (Park et al., 1996).The best crystals were obtained at 291 K using the hanging-dropmethod by mixing 1 ml protein solution containing 5 mM ATP or50 mM ADP and 50 mM d-alanyl-d-alanine with 1 ml crystallizationsolution containing 0.2M MgCl2, 25% PEG 3350, 0.1M Tris–HClpH 8.0 and equilibrating against a 500 ml reservoir. Crystals appearedafter 2 d as plates of dimensions of 0.1  0.35 mm (Figs. 2 and 3). Weare confident these crystals contained the ligands that were cocrys-tallized since EcDdlB did not crystallize under the same conditions inthe apo form or with ATP, ADP or d-alanyl-d-alanine alone.crystallization communications406 Batson et al.  D-Alanyl-D-alanine ligase Acta Cryst. (2010). F66, 405–408Figure 112% SDS–PAGE analysis of the purification of EcDdlB. LaneMr, molecular-weightstandards (kDa); lane 1, crude cell lysate; lane 2, flowthrough from immobilizedmetal-affinity chromatography; lane 3, peak from immobilized metal-affinitychromatography; lane 4, peak from size-exclusion chromatography.Initial diffraction experiments were carried out at the ESRF, butcomplete data sets were collected to resolutions of 1.4 A˚ for EcDdlB–ATP–d-alanyl-d-alanine and 1.5 A˚ for EcDdlB–ADP–d-alanyl-d-alanine on the I04 beamline at the Diamond synchrotron (UK)using an ADSC Q315 CCD detector with an oscillation angle of 1and a crystal-to-detector distance of 160 mm. All data were indexed,integrated and scaled using the HKL suite of programs (Otwinowski& Minor, 1997). Data-collection and processing statistics are shownin Table 1. For each data set, Matthews probability calculationssuggested the presence of two molecules in the asymmetric unit,although a self-rotation function did not reveal the presence of anoncrystallographic rotational axis (Matthews, 1968; Kantardjieff &Rupp, 2003). A native Patterson map suggested the presence ofpseudo-translation, with a high peak in the (u, v, 12) Harker section(94 and 31, corresponding to 40% and 13% of the origin-peakheight, for the two data sets, respectively). Molecular-replacementsolution using Phaser (McCoy et al., 2007) with the coordinates of theY216F EcDdlB mutant (PDB code 1i0w; Fan et al., 1997) as a modelsubsequently revealed two molecules in the crystallographic asym-metric unit which are related to each other by a twofold rotationparallel to the z crystallographic axis for both data sets.Models of the EcDdlB–ATP–d-alanyl-d-alanine complex and theEcDdlB–ADP-d-alanyl-d-alanine complex are currently being builtand refined. We are also seeking to crystallize the protein in the apoform and in complex with inhibitors. Ultimately, these studies maycontribute to further understanding of the individual reaction steps ofthe mechanism, the activity-associated conformational changes andthe rational design of specific inhibitors against Ddls.This work was supported by a University of Warwick PhDstudentship to SB and in part by MRC research grants G500643 andG0600801 and Wellcome Trust equipment grants 071998 and 068598.Equipment used in this research was obtained through theBirmingham–Warwick Science City Translational Medicine: Experi-mental Medicine Network of Excellence project, with support fromAdvantage West Midlands (AWM). Crystallographic data werecollected at beamlines IO4 at Diamond Light Source, UK and atID14.1 at the ESRF, France, and we acknowledge the support ofbeamline scientists Ralf Flaig (Diamond) and Matthew Bowler(ESRF).ReferencesBugg, T. D., Dutka-Malen, S., Arthur, M., Courvalin, P. & Walsh, C. T. (1991).Biochemistry, 30, 2017–2021.Daub, E., Zawadzke, L. E., Botstein, D. & Walsh, C. T. (1988). Biochemistry,27, 3701–3708.Fan, C., Moews, P. C., Walsh, C. T. & Knox, J. R. (1994). Science, 266, 439–443.Fan, C., Park, I. S., Walsh, C. T. & Knox, J. R. (1997). Biochemistry, 36, 2531–2538.Kantardjieff, K. A. & Rupp, B. (2003). Protein Sci. 12, 1865–1871.Kitamura, Y., Ebihara, A., Agari, Y., Shinkai, A., Hirotsu, K. & Kuramitsu, S.(2009). Acta Cryst. D65, 1098–1106.Kuzin, A. P., Sun, T., Jorczak-Baillass, J., Healy, V. L., Walsh, C. T. & Knox,J. R. (2000). Structure, 8, 463–470.Lee, J. H., Na, Y., Song, H. E., Kim, D., Park, B. H., Rho, S. H., Im, Y. J., Kim,M. K., Kang, G. B., Lee, D. S. & Eom, S. H. (2006). Proteins, 64, 1078–1082.Lessard, I. A., Healy, V. L., Park, I. S. & Walsh, C. T. (1999). Biochemistry, 38,14006–14022.Liu, S., Chang, J. S., Herberg, J. T., Horng, M. M., Tomich, P. K., Lin, A. H. &Marotti, K. R. (2006). Proc. Natl Acad. Sci. USA, 103, 15178–15183.Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497.McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni,L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658–674.Mullins, L. S., Zawadzke, L. E., Walsh, C. T. & Raushel, F. M. (1990). J. Biol.Chem. 265, 8993–8998.Neuhaus, F. C. (1962). J. Biol. Chem. 237, 778–786.Neuhaus, F. C. & Lynch, J. L. (1962). Biochem. Biophys. Res. Commun. 8,377–382.crystallization communicationsActa Cryst. (2010). F66, 405–408 Batson et al.  D-Alanyl-D-alanine ligase 407Figure 3Photograph of EcDdlB crystals obtained in the presence of ADP and d-alanyl-d-alanine grown in 0.2M MgCl2, 25% PEG 3350, 0.1M Tris–HCl pH 8.0. Theapproximate dimensions of the individual crystals are 0.1  0.4  0.02 mm.Table 1Data-collection and processing statistics.Values in parentheses are for the highest resolution shell.EcDdlB–ATP–d-alanyl-d-alanineEcDdlB–ADP–d-alanyl-d-alanineBeamline I04, Diamond I04, DiamondDetector ADSC Q315 CCD ADSC Q315 CCDWavelength (A˚) 0.9763 0.9763Space group P212121 P212121Unit-cell parametersa (A˚) 51.23 53.00b (A˚) 97.80 97.62c (A˚) 110.12 109.49Molecules per ASU 2 2Matthews coefficient (A˚3 Da1) 2.09 2.14Solvent convent (%) 41 43Resolution range (A˚) 49–1.4 (1.45–1.4) 48–1.5 (1.55–1.5)Total observations 648917 601481Unique reflections 106197 91443Average I/(I) 22.4 (2.1) 18.9 (2.1)Rmerge† (%) 0.069 (0.577) 0.093 (0.578)Completeness (%) 97.1 (94.6) 99.9 (99.1)† Rmerge =PhklPi jIiðhklÞ  hIðhklÞij=PhklPi IiðhklÞ, where Ii(hkl) is the ith observa-tion of reflection hkl and hI(hkl)i is the mean intensity of that reflectionFigure 2Photograph of EcDdlB crystals obtained in the presence of ATP and d-alanyl-d-alanine grown in 0.2M MgCl2, 25% PEG 3350, 0.1M Tris–HCl pH 8.0. 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Biochemistry, 30, 1673–1682.crystallization communications408 Batson et al.  D-Alanyl-D-alanine ligase Acta Cryst. (2010). F66, 405–408

crystallization communications Acta Cryst.

Crystallization and preliminary X-ray analysis of a D-alanyl-D-alanine ligase (EcDdlB) from Escherichia coli

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Crystallization and preliminary X-ray analysis of a D-alanyl-D-alanine ligase (EcDdlB) from Escherichia coli

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